Skeletal muscle-associated toxicity is an underresearched area in the field of high-throughput toxicity screening; hence, the potential adverse effects of drugs and chemicals on skeletal muscle are largely unknown. Novel organotypic microphysiological in vitro models are being developed to replicate the contractile function of skeletal muscle; however, the throughput and a need for specialized equipment may limit the utility of these tissue chip models for screening. In addition, recent developments in stem cell biology have resulted in the generation of induced pluripotent stem cell (iPSC)-derived skeletal myoblasts that enable high-throughput in vitro screening. This study set out to develop a high-throughput multiplexed assay using iPSC-derived skeletal myoblasts that can be used as a first-pass screen to assess the potential for chemicals to affect skeletal muscle. We found that cytotoxicity and cytoskeletal integrity are most useful and reproducible assays for the skeletal myoblasts when evaluating overall cellular health or gauging disruptions in actin polymerization following 24 h of exposure. Both assays are based on high-content imaging and quantitative image processing to derive quantitative phenotypes. Both assays showed good to excellent assay robustness and reproducibility measured by interplate and interday replicability, coefficients of variation of negative controls, and Z′-factors for positive control chemicals. Concentration response assessment of muscle-related toxicants showed specificity of the observed effects compared to the general cytotoxicity. Overall, this study establishes a high-throughput multiplexed assay using skeletal myoblasts that may be used for screening and prioritization of chemicals for more complex tissue chip-based and in vivo evaluation.