Skeletal muscle is an intricately structured tissue made up of a complex framework of various cell types. The dynamic spatial and temporal relationships among these cells during both homeostasis and periods of injury contribute to the regenerative abilities of skeletal muscle. Currently, there is a deficiency in quantitative assessment, biological role, and the molecular mechanisms that could elucidate a possible juxtavascular niche for muscle satellite cells, a stem cell population for skeletal muscle regeneration. To fully comprehend the regeneration process by muscle satellite cells, a three-dimensional (3-D) imaging approach is essential. Confocal microscopy serves as an exceptional method for examining the spatial arrangement of cells within a specific tissue. In this protocol, we provide a detailed procedure for preparing optically transparent extensor digitorum longus (EDL) skeletal muscle specimens that are appropriate for confocal microscopy and computational 3-D assessment. We outline the steps for sample preparation, which include perfusion fixation and the tissue clearing process for rodent muscle specimens, as well as guidelines for image capture and computational evaluation featuring sample segmentation and 3-D visualization. This methodology can be utilized to characterize diverse cell types, such as muscle satellite cells and capillary endothelial cells found in rodent skeletal muscle.