Mitochondrial morphology and membrane potential (Δψ) are important readouts of mitochondrial function. Integrated analysis of these parameters in living cells can be performed using fluorescent lipophilic cations, which enter cells and accumulate in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of tetramethylrhodamine methyl ester (TMRM) and Mitotracker Green FM (MG) for mitochondrial morphology and semiquantitative Δψ analysis in living primary human skin fibroblasts (PHSFs). Practically, we present an integrated protocol to quantify mitochondrial morphology parameters and signal intensity using epifluorescence microscopy of PHSFs co-stained with TMRM and MG. This approach performs best using large flat cells like PHSFs, which display a high mitochondria-specific fluorescence signal and are imaged at a relatively high (x40) magnification.