Mitochondria play a vital role in the regulation of intracellular calcium dynamics. Fluorescent dyes can be used to provide a direct measurement of the redox state, mitochondrial membrane potential, and mitochondrial calcium content. The simplicity of this approach lends itself to high-throughput assays and time-resolved analyses; however, care must be taken to avoid artifactual results. We outline general methods using confocal microscopy for analysis of the redox state, mitochondrial membrane potential, and mitochondrial calcium content in adult cardiomyocytes. We demonstrate how these parameters can be analyzed in parallel using the emission spectra "fingerprinting" method even when emission spectra overlap.