Hydroxyproline (Hyp) is an imino acid posttranslationally formed by sequence-specific hydroxylases in the repeating collagen Gly-Xaa-Yaa triad present in all collagen types of all species. In both Xaa- and Yaa-positions, Pro is the most common residue, often oxidized to 4-Hyp in the Yaa- and rarely to 3-Hyp in the Xaa-positions. Here we describe the qualitative and quantitative analysis of 3- and 4-Hyp-isomers by separating the free imino acids either with hydrophilic interaction chromatography (HILIC) or after derivatization with reversed-phase chromatography (RPC). In both cases the compounds were detected by electrospray-ionization mass spectrometry.