Collagen/gelatin fragments are often polydisperse and heterogeneous, making them difficult for quantitation, especially during in vivo circulation. In this study, a new quantitation method was established based on the detection of collagen-specific peptide markers using HPLC-MS/MS. Blood samples were collected and digested for collagen-specific peptides identification and content determination after the infusion of bovine gelatin fragments into rabbits. Thirteen collagen-specific peptides, of which five were present in bovine collagen but absent in human collagen, were identified by MS detection and could be used as peptide markers for the quantitation of gelatin fragments in blood. One of the bovine collagen specific-peptides was then used as a peptide marker, and its detection linearity range and limit of quantification were confirmed. The pharmacokinetic parameters, such as half-life and elimination rate, and also the pharmacokinetic model of the infused gelatin fragments were determined from the time-concentration curve. This peptide marker based method offers better selectivity towards collagen/gelatin fragments quantitation and could find wider applications in the field of collagen/gelatin based products such as drug delivery materials, and nanoparticles.