Here, we provide a protocol for isolation of mouse primary skeletal muscle fibers using two alternative approaches—enzymatic dissociation or mechanical microdissection. We describe the procedures for surgical removal of muscle of interest and isolation of intact single-muscle fibers by either collagenase digestion or mechanical microdissection. We then detail intracellular calcium measurements by microinjecting or loading the isolated muscle fibers with membrane permeable calcium dyes. Finally, we outline steps for intracellular calcium quantification by fluorescent measurement. For complete details on the use and execution of this protocol, please refer to Gineste et al.1 Display omitted •Enzymatic dissociation or mechanical microdissection to isolate mouse muscle fibers•Microinject or load isolated muscle fibers with membrane permeable calcium dyes•Steps for intracellular calcium quantification by fluorescent measurement•Microdissection preserves fiber microenvironment and maintains phenotypes and functions Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we provide a protocol for isolation of mouse primary skeletal muscle fibers using two alternative approaches—enzymatic dissociation or mechanical microdissection. We describe the procedures for surgical removal of muscle of interest and isolation of intact single-muscle fibers by either collagenase digestion or mechanical microdissection. We then detail intracellular calcium measurements by microinjecting or loading the isolated muscle fibers with membrane permeable calcium dyes. Finally, we outline steps for intracellular calcium quantification by fluorescent measurement.