The purpose of this study was to measure metabolic changes in mesenchymal stem cells (MSCs) placed in osteogenic medium by autofluorescence spectroscopy. MSCs were plated in stem cell-supporting or osteogenic medium and imaged. Shift from the basic growth environment to the inductive osteogenic environment was confirmed by reverse transcription-polymerase chain reaction. Reduced pyridine nucleotides were detected by exciting near 366 nm and measuring fluorescence at 450 nm, and oxidized flavoproteins were detected by exciting at 460 nm and measuring fluorescence at 540 nm. The ratio of these fluorescence measurements, reduction-oxidation (redox) fluorometry, is a noninvasive measure of the cellular metabolic state. The detected pyridine nucleotide to flavoprotein ratio decreased upon transitioning from the stem cell to the differentiated state, as well as with increasing cell density and cell-cell contact. MSC metabolism increased upon placement in differentiating medium and with increasing cell density and contact. Redox fluorometry is a feasible, noninvasive technique for distinguishing MSCs from further differentiated cells.