Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology.