Background: Patients with Duchenne Muscular Dystrophy (DMD) have gait disorders. Therefore, specific gait assessment tools are needed. Aims: The aim of this study was to develop a gait assessment instrument for DMD patients (DMD-GAS), and investigate its validity and reliability. Study design: The scale was developed considering the expert opinions which included 10 physiotherapists who had experience in the management of patients with DMD, and the Content Validity Index (CVI) was calculated. The final version of the DMD-GAS that was agreed upon the experts consisted of 10 items, and each item scored between 0 and 2. The intra-rater reliability was established by the video analysis of children with a 1-month interval and inter-rater reliability was determined by the scores of 3 physiotherapists. Subjects: The study included 56 patients with DMD. Outcome measures: The criterion validity was determined by investigating the relationship between the total score of the DMD-GAS and Motor Function Measure (MFM), 6 Minute Walk Test (6MWT), and the data obtained from GAITRite. Results: The CVI of the DMD-GAS was 0.90 (p < 0.05). The construct validity and internal consistency of the DMD-GAS were excellent as well as the intra- and inter-rater reliability (>0.90). Moderate-to-very strong correlations were found between the total score of the DMD-GAS and the MFM-total score (r = 0.78), 6MWT (r = 0.71), gait speed (r = 0.50), stride length (r = 0.56), and base of support (r = −0.70) (p < 0.01). Conclusions: The results indicated that DMD-GAS was a reliable and valid instrument to determine gait characteristics of the patients with DMD in clinical settings. Clinical trial number: NCT05244395.