Background: Green leafy vegetables are nutritionally valuable sources of the carotenoids lutein and β-carotene, which are thought to have potential beneficial health effects on different aspects of vision. Bioavailability of these compounds is low, however, and depends on a complex set of factors, which could potentially influence populations at high risk of malabsorption. The bioavailability potential is difficult to investigate in vivo, therefore, alternative in vitro approaches are currently being applied for investigating in vitro accessibility as potential estimates of the bioavailability potential. The usefulness of these results is, however, limited by the lack of validated in vitro-in vivo results. Aims: The present PhD thesis investigates liberation and in vitro accessibility of the carotenoids lutein and β-carotene following domestic kitchen preparation procedures for green leafy vegetables of different cultivars. The aim was furthermore to test the validity of in vitro accessibility as a possible predictor of the bioavailability of carotenoids from green leafy vegetables in healthy subjects and in patients with surgically altered gut absorption. Methods: Influence of cultivar type and domestic kitchen procedures on liberation and in vitro accessibility of carotenoids from green leafy vegetables was investigated in a modified in vitro digestion method. Comparability between in vitro accessibility and the bioavailability potential were studied in a combined in vitro digestion-in vivo cross-over, controlled postprandial and short-term follow-up validation trial in both healthy individuals and short bowel patients. Moreover, an ultra-high performance liquid chromatography and photo-diode array detector coupled method was developed and applied for quantification of carotenoids from green leafy vegetables, faeces, serum and chylomicrons. Results: In vitro digestion: Particle size reduction through mincing and pureeing was shown to be the most important influential factor for Lut and β-Car liberation and in vitro accessibility from green leafy vegetables. Additionally, fat addition to pureed spinach increased liberation (p < 0.05) but not in vitro accessibility of β-Car and had no or a negative effect on Lut liberation and in vitro accessibility. No differences were observed between the different fat types on the level of in vitro accessibility. The degree of heating had variable effects on carotenoid liberation from different cultivars of Asia salads and negative or no effects on liberation and in vitro accessibility of carotenoids from spinach. In vitro-in vivo study: Mincing resulted in a factor two difference in in vitro accessibility of carotenoids when comparing whole leaf and minced spinach after in vitro digestion. In healthy subjects, this difference resulted in significantly higher β-Car concentrations in the triglyceride rich lipoprotein fractions (TLR)/Chylomicron (CH) fraction at 4 and 5 hours postprandially (p < 0.05) after intake of minced spinach compared to whole leaf spinach, but did not differ over time in the two-week follow-up. No difference in carotenoid response was found in short bowel patients either postprandially or in the short-term follow-up period. Moreover, an improved ultra-high-performance liquid chromatography (UHPLC) - diode array detector (DAD) based method was developed and validated for quantification of major carotenoids present in green leafy vegetables, serum, chylomicrons and faeces. Conclusions: Both in vitro and in vivo methodologies show strengths and limitations when evaluating the in vitro accessibility potential and accordingly bioavailability of carotenoids. The current findings, following digestion of green leafy vegetables, demonstrated a significant increase in both liberation and in vitro accessibility of Lut and β-Car after plant cell disintegration due to the degree of mincing in spinach. The degree of heating had variable positive effects on carotenoid liberation from different cultivars of Asia salads and negative or no effects on liberation and in vitro accessibility of carotenoids from spinach. Similarly, fat addition influenced β-car liberation positively; however, the effect was eliminated on the level of in vitro accessibility. In vitro accessibility of carotenoids following in vitro digestion of spinach could possibly be a predictor for the bioavailability potential of β-Car in healthy subjects, but not for Lut. The difference in bioavailability for Lut and β-Car between the two matrices was not reflected in the carotenoid response in short bowel patients.