The amino-terminal propeptide of type III procollagen (P-III-NP) is used with IGF-I to detect the illicit use of growth hormone and to monitor growth hormone therapy. However, the only currently available assays for P-III-NP are immunoassays, which are not well harmonized. In addition, other fragments of type III procollagen may better evaluate collagen turnover. We developed a high-throughput assay using immunoaffinity enrichment coupled to ultra high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify peptides belonging to three different regions of type III procollagen in human serum simultaneously.