Oxidative stress underlies the mechanisms of many diseases such as Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease, schizophrenia, diabetes, Multiple Sclerosis, bipolar disorder, and chronic fatigue syndrome. Oxidative stress is caused by excessive reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals, which are byproducts of oxygen metabolism in aerobic organisms. Environmental factors such as exposure to ultraviolet light, chemicals ingested by the diet, ionizing radiation, and cigarette smoke can also lead to production of ROS and therefore oxidative stress. As a result, there is a growing need for quantitative and noninvasive methodologies to probe oxidative stress at the single-cell level and ultimately in vivo. In this thesis, we examined the potential of natural coenzyme nicotinamide adenine dinucleotide (NADH) as a natural biomarker for oxidative stress in C3H 10T1/2 living cells in culture. NADH (fluorescent) is a coenzyme that is essential for energy metabolism via oxidative phosphorylation pathway in the inner membrane of mitochondria, which is also a major source for intrinsic ROS species generated through the electron transport chain. Our experimental multiparametric approach combined cell culture with fluorescence microscopy (confocal and two-photon) and spectroscopy (fluorescence lifetime imaging and time-resolved anisotropy) methods. Cultured cells were treated with hydrogen peroxide and rotenone in order to trigger oxidative stress. As a point of reference, conventional oxidative stress assays such as MitoSOX Red, JC-1, and H2DCFDA were used to optimize the chemical dosage and incubation time needed for observable oxidative stress. Our results help in the collective effort to establish cellular NADH autofluorescence as a natural biomarker for cellular metabolic health and oxidative stress.