Background and Objective The photobiological effect of laser light on cells and tissues originates from light absorption by endogenous chromophores and hence it depends on the wavelength of light source and cell type. Earlier studies regarding the biostimulation effects of green laser light investigated a wide variety of cells but not adipose tissue-derived stem cells (ADSCS). In this study we reported the in vitro effect of 532-nm Nd:YAG laser on proliferation, mitochondrial activity of these mesenchymal stem cells (MSCs) on the autofluorescence emission at wavelengths associated with nicotinamide adenine dinucleotide (NADH) and flavoproteins. Materials and Methods ADSCS were exposed to 532 nm second harmonic generation laser light at moderate power density (0.153 W/cm2) for periods of 30, 45, 60, 180, and 300 seconds. Mitochondrial membrane potential was measured using JC1 stain and confocal laser scanning microscopy, cell proliferation rates, and cellular autofluorescence emission at 450 and 540 nm wavelengths were measured using micro plate spectrofluorometer 48 hours after irradiation. Results Shorter (30-60 seconds) exposure times led to significantly increased proliferation, attributed to increased mitochondrial activity (P < 0.05). At longer exposures we observed a significant decrease in proliferation and autofluorescence (P < 0.05). Strong correlation was observed between proliferation rates of cells and autofluorescence intensity. Conclusion Our results show that autofluorescence of the respiratory chain components and key autofluorescent metabolites offers a non-invasive method to quantify cellular response to laser irradiation. Lasers Surg. Med. 44: 769-778, 2012.