Loss of basement membrane (BM) integrity is typically associated with cancer. Nidogen-1 is an essential component of the BM. Nidogen-1 is a substrate for cathepsin-S (CatS) which is released into the tumor microenvironment. Measuring nidogen-1 degraded by CatS may therefore have biomarker potential in cancer. The aim of this study was to investigate if CatS-degraded nidogen-1 was detectable in serum and a possible biomarker for cancer, a pathology associated with disruption of the BM. A competitive enzyme-linked immunosorbent assay (NIC) was developed with a monoclonal mouse antibody specific for a CatS cleavage site on human nidogen-1. Dilution and spiking recovery, inter- and intravariation, as well as accuracy were evaluated. Serum levels were evaluated in patients with breast cancer, small cell lung cancer (SCLC), and non-SCLC (NSCLC) and in healthy controls. The results indicated that the NIC assay was specific for nidogen-1 cleaved by CatS. Inter- and intraassay variations were 9% and 14%, respectively. NIC was elevated in NSCLC as compared to healthy controls (P < .001), breast cancer (P < .01), and SCLC (P < .5). The diagnostic power (area under the receiver operating characteristics) of NIC for NSCLC as compared to all other samples combined was 0.83 (95% confidence interval: 0.71-0.95), P < .0001. In conclusion, nidogen-1 degraded by CatS can be quantified in serum by the NIC assay. The current data strongly suggest that cathepsin-S degradation of nidogen-1 is strongly associated with NSCLC, which needs validation in larger clinical cohorts.