Rheumatic joints have an altered cartilage turnover. Cartilage intermediate layer protein 1 (CILP-1) is secreted from articular chondrocytes and deposited into the cartilage extracellular matrix. We developed an immunoassay targeting a Matrix Metalloproteinase (MMP)-generated neo-epitope of CILP-1, named CILP-M. Human articular cartilage was cleaved with proteolytic enzymes and CILP-M levels were measured. We also quantified CILP-M in two studies from patients with rheumatoid arthritis (RA), ankylosing spondylitis (AS) and osteoarthritis (OA) and explored the monitoring and prognostic potential of CILP-M in TNF-α inhibitory treatment and modified Stoke AS Spine Score (mSASSS) progression. CILP-M was generated by MMP-1, -8 and -12. In the discovery study, CILP-M was significantly higher in patients with RA, AS and OA than healthy donors (p < 0.01, p < 0.001, p < 0.05) with an area under the curve (AUC) between the diseased groups and healthy donors > 0.95 (p < 0.001). In the validation study, patients with RA and AS had significantly higher CILP-M levels than healthy controls (p < 0.001) and AUC > 0.90 (p < 0.001). Patients with AS treated with TNF- α inhibitory treatment in the validation study had significantly lower CILP-M levels after treatment (p = 0.004). CILP-M may provide useful insights into cartilage degradation processes in rheumatic diseases.