An analytical method for determining seleno-methionine (SeMet), methyl-seleno-cysteine and seleno-cystine in extra-virgin olive oil (EVOO) was developed and validated. EVOO sample (15 g) was diluted with hexane, extracted with methanol/water 80:20 (v/v), and cleaned up by a reversed phase/strong cation exchange solid phase extraction. Analysis was performed by chiral hydrophilic interaction liquid chromatography-tandem mass spectrometry. Process efficiency ranged between 49 and 97% and trueness between 87 and 126%, with intermediate precision, expressed as standard deviation, lower than 10%. Method detection limits (MDLs) and method quantification limits (MQLs) were lower than 1 μg kg⁻¹. Thirty-two EVOO samples from different Italian regions were analyzed for both total Se and single seleno-amino acids determination. Only L-SeMet was found at level MQL (0.2 μg kg⁻¹)-1.42 μg kg⁻¹ in ten samples, while total Se was in the range of MDL-9.1 μg kg⁻¹. Concentration of L-SeMet (5–6% of total Se) and total Se correlated very well to each other (R² = 0.995).