We propose a novel approach to assess simultaneously lipid composition in lipid droplets, the redox state of cytochromes, and the relative amount of [Fe–S] clusters in the electron transport chain in the mitochondria of skeletal myocytes by means of near-infrared Raman spectroscopy. Mitochondria dysfunction, disruption of oxidative phosphorylation, and change in lipid metabolism may be a consequence of the obesity-related insulin resistance and diabetes mellitus. Lipid metabolism affects the state of the mitochondrial electron transport chain although these relations are not well understood. We demonstrate the applicability of the proposed approach in a case study of myocytes of an obese patient before and after the gastric bypass surgery in comparison with a healthy lean donor. Ratios from chosen Raman peaks were calculated and compared between the different subjects. We show that the suggested technique allows to estimate qualitatively the relative amount of cholesterol and unsaturated lipids, ordering of lipid phase in lipid droplets, changes in the redox state of c-type and b-type cytochromes, and the relative amount of [Fe–S] clusters in the mitochondria of intact myocytes. The proposed approach opens perspectives to uncover relations between the functional state of mitochondrial cytochromes and lipid composition and to develop a monitoring tool once it is applied to a wide cohort of patients.