Autoantibodies against melanoma differentiation-associated gene 5 (MDA-5) are one of the serological markers for DM. Anti-MDA-5 antibodies are especially associated with rapidly progressive interstitial lung disease (ILD) in amyopathic DM (ADM). It is known that the antibody status of anti-ENAs does not generally change significantly with disease course. For anti-MDA-5 antibodies, however, few longitudinal studies have investigated such changes. This study aimed to establish a quantitative assay for anti-MDA-5 antibodies towards assessing the long-term outcome of ADM patients who had anti-MDA-5 antibodies. We established ELISA for measuring anti-MDA-5 antibody levels using in vitro transcription and translation recombinant protein. The antibody levels were measured at different time points in 11 clinically ADM patients who tested positive for the anti-MDA-5 antibody on their first visit (range of follow-up 3 months to 16 years). At the stage of clinical remission, six patients received no medication and the four others received low-dose CS. ELISA showed that anti-MDA-5 antibodies disappeared in nine of the patients and fell to just above the cut-off in one patient; in the patient who died, the antibodies remained. Our results suggest that anti-MDA-5 antibodies may be useful as a marker for monitoring disease activity in ILD complicated with ADM. Serial monitoring at short intervals is required to evaluate whether anti-MDA-5 antibody levels correlate with ADM disease activity.