OBJECTIVES: To evaluate the performance of a commercial line immunoassay (LIA) in cohorts of healthy controls (HC), disease controls (DC), and patients with idiopathic inflammatory myopathies (IIMs), and investigate whether different cut-offs would enhance the accuracy of myositis antibodies (MAs) by LIA. METHODS: Sera from patients with positive MAs (n = 118), DC (n = 124) and HC (n = 60), were analysed for MAs using EUROLINE Myopathies 16 Ag LIA (EUROIMMUN). RESULTS: The newly established MAs cut-offs resulted in significantly lower both false-positive and multiple positive rates compared with the manufacturer's single cut-off, from 36.7% to 5.0% and 7.2% to 1.1% in controls, and 45.8% to 23.8% and 23.7% to 15.5% in patients with positive MAs cohorts, respectively. Furthermore, the positive predictive value (PPV) of MAs for IIMs or interstitial lung disease (ILD) increased significantly from 47.5% to 66.7%. CONCLUSION: Establishment of local cut-offs and cross-reference ANA findings can significantly enhance the assay's performance and increase the confidence in interpreting MAs results by LIA. It is important to be aware of the low PPV for most MAs in real-world cohorts, thus, the investigation of MAs should be approached as confirmatory or differential diagnosis rather than a screening tool in IIMs.