Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time-resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC-5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue- and red-shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra-cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.