Advanced glycation end products (AGEs) and protein cross-links have been extensively investigated in both food and biomedical fields over the past years. Although there are a few chromatographic and immunological methods for the analysis of selected AGEs, there is no method available for comprehensive simultaneous analysis of major AGEs found in processed foods and biological samples. In the present study, we have reported a validated UHPLC-MS/MS method for simultaneous identification and quantification of 15 different AGEs, furosine (an indicator of Amadori products), 2 protein-derived cross-links (lanthionine and lysinoalanine) and 2 amino acids (Lys and Arg). The analytes were separated on a reversed phase C-18 column and quantified accurately based on the isotope dilution method, where 9 stable isotope-labelled internal standards were used to quantify 20 different analytes using an Orbitrap mass analyzer. The method showed acceptable linearity, accuracy and precision. The LOD and LOQ values in plasma were in the range of 0.30–19.02 and 0.87–57.06 ng/mL, respectively. The recovery values at the three spiked levels were in the range of 71–110%, with some exceptions. The intraday and interday precision were in the range of 1.5–13.2%, however, quantification of N-ɛ-(carboxymethyl)lysine accompanied slightly higher interday precision (30.7%). The applicability of the method was successfully assessed by analyzing AGEs and protein cross-links in six different complex matrices including Ultra-High Temperature (UHT) processed milk, roasted chicken breast meat, roasted chicken skin, roasted pork liver, bovine plasma and perfusion liquid.