The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP). This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab) was raised in mouse, targeting specifically PIIBNP (QDVRQPG) and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6-2.2 nM), human amniotic fluid (163-188 nM) and sera from different animal species, e.g., fetal bovine serum (851-901 nM) with general good linearity (100% (SD 7.6) recovery) and good intra- and inter-assay variation (CV% < 10). Dose (0.1 to 100 ng/mL) and time (7, 14 and 21 days) dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX) and human cartilage explants (HEX) upon stimulation with insulin-like growth factor (IGF-1), transforming growth factor (TGF)-β1 and fibroblastic growth factor-2 (FGF-2). TGF-β1 and IGF-1 in concentrations of 10-100 ng/mL significantly (p < 0.05) induced release of PIIBNP in BEX compared to conditions without treatment (WO). In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.