BACKGROUND: The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. METHODS: We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. RESULTS: The assay was linear over an estimated concentration range of 0.3 to1.0 nM and 0.1 to 0.4 nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. CONCLUSIONS: Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.