Objective To compare specificity and sensitivity of a commercially available fixed cell-based assay (F-CBA) to radioimmunoprecipitation assay (RIPA) for acetylcholine receptor antibody (anti-AChR) detection in myasthenia gravis (MG). Background Approximately 50% of ocular and 85% of generalized MG are anti-AChR positive by RIPA, the current gold standard test. Clustered live cell-based assay (L-CBA) can detect low-affinity anti-AChR that are missed by RIPA, but the costly and time-consuming nature of L-CBA has restricted its use to specialized centres. A commercial F-CBA has become available for anti-AChR detection, but its diagnostic performance compared to RIPA requires evaluation. Design/Methods In this retrospective diagnostic cohort study we reviewed the clinical information of suspected MG patients evaluated at London Health Sciences Centre MG clinic, who were clinically classified as MG or non-MG and who had anti-AChR RIPA and then F-CBA performed. Classification of each patient as anti-AChR F-CBA-negative/positive, RIPA-negative/positive, and MG/non-MG permitted specificity and sensitivity calculations for each assay. Results Six-hundred-eighteen patients were included in study analysis. The median patient age at time of sample collection was 45.8 years (range: 7.5–87.5 years) and 312/618 (50.5%) were female. Of 618 patients, 395 (63.9%) were classified as MG. Specificity of both F-CBA and RIPA was excellent (99.6% vs. 100%, P > 0.99). One F-CBA-positive patient was classified as non-MG, although in retrospect ocular MG with functional overlay was challenging to exclude. Sensitivity of F-CBA was significantly higher than RIPA (76.7% vs. 72.7%, P = 0.002). Overall, 20/97 (21%) otherwise SNMG patients after RIPA evaluation had anti-AChR detected by F-CBA. Conclusions In our study anti-AChR F-CBA and RIPA both had excellent specificity, while F-CBA had 4% higher sensitivity for MG and detected anti-AChR in 21% of SNMG patients. Our findings indicate that F-CBA is a viable alternative to RIPA for anti-AChR detection.