A generic method for the quantification of type II collagen in protein-based dietary supplements is described. This quantitative analysis was conducted using liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-ESI-TOF MS). Compared to classical methods with the use of isotope-labeled standards, our method includes, for the first time, the quantification of hydroxyproline using histidine as an internal standard. Separation of the analytes was performed on a Phenomenex Synergi 4 mu m Fusion-RP 80 C-0 column (150 x 2.0 mm, 4.0 mu m) with a mobile phase made of 10 mM ammonium formate in water (A) and 10 mM ammonium formate in methanol (B). The assay was fully validated according to FDA guidelines, and the method exhibited sufficient specificity, accuracy, and precision. Intra- and inter-batch accuracy, determined as a deviation between nominal and measured values, ranged from -4.8 to 9.1% and from 0.9 to 6.4 %, respectively. All analytes (hydroxyproline and histidine) at three concentration levels showed extraction recoveries from 89 to 98 %. The method was successfully applied to protein-based dietary supplements of the pharmaceutical industry.