We propose a simple method to fix, attach and premeabilize isolated mouse cardiomyocytes for immuno-staining and confocal microscopic imaging. This method is highly reproducible and retains the morphology of the cardiomyocytes. This method will be useful to study morphological changes in the cytoskeletal protein architecture, organellar and protein distribution within the cell without any distortion of intracellular organization. This protocol can be completed in less than 2 days.