Skeletal muscle function and quality are strong indicators of metabolic health. Here, we present a protocol for skeletal muscle phenotyping in zebrafish. We describe steps to evaluate spontaneous locomotion, measure oxygen consumption during incremental exercise, and analyze cross-sectional area of skeletal muscle cells in adult zebrafish. This protocol has potential applications to assess zebrafish muscle quality and function in studies of metabolic diseases, aging, and skeletal muscle health. For complete details on the use and execution of this protocol, please refer to Grepper and Tabasso et al. ¹ .