Autofluorescence (AF) is the natural emission of light by intrinsic fluorophores. Oxidized mitochondrial flavins, lipofuscin and reduced nicotinamideadenine dinucleotide phosphate (NAD(P)H) are the main sources of the autofluorescence in cells upon excitation with visible light. The aim of the study was to investigate changes in the metabolism of four cell lines by monitoring their autofluorescence with a microplate reader. Autofluorescence intensities of cells were collected at two wavelengths for the excitation and fluorescence emission: for endogenous NAD(P)H at 366/450 nm, for the oxidized flavoproteins and lipofuscin at 460/540 nm. Human mesenchymal stem cells (hMSC), epithelial cells from mammary gland (MCF 10A), breast ductal carcinoma (T-47D) prostate carcinoma (DU-145) were observed daily for 16 days. The level of NAD(P)H autofluorescence did not differ among the cell lines investigated. The significant increase in oxidized flavoproteins fluorescence intensity was recorded for hMSC and ranged from 140 to 175% of control. During 28 days differentiation process, the NAD(P)H, FAD and lipofuscin fluorescence intensities were recorded every day, and the redox ratio was then calculated. The redox ratio gradually decreased during the last eight days of osteogenesis and adipogenesis. Therefore, in our opinion the NAD(P)H, FAD, and lipofuscin fluorescence emission at the wavelengths selected are the optimal parameters to be collected during the differentiation process in order to monitor the metabolism of hMSC undergoing structural and morphological changes.