 The study aimed to isolate purified mitochondria from mouse skeletal muscle using percol density gradient, assess bioenergetic function and purity via seahorse extracellular flux, XF, analyses and mass spectrometry. Results showed that the method yielded 200 to 400 UG protein with optimum mitochondrial input at 500-ing, respiratory control ratio ranging from 3.9 to 7.1 using various substrates, and significant enrichment of mitochondrial proteins indicating high sample purity. The study established a methodology that ensures sufficient high-quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics. This article was authored by Rhea Victoria P. Anansiada-Cosa, Anjanya Akanta, Calvin P. Vary, and others.