Photobiomodulation (PBM) a promising non-invasive therapeutic technique has been proposed to have regenerative ability. There is limited understanding of PBM effectiveness in 3D cell culture systems. To evaluate the effects of PBM at 825 nm on adipose-derived stem cells (ADSCs) cultured in 2D and 3D spheroid models. ADSCs were exposed to PBM at fluences of 5, 10, and 15 J/cm 2 to assess cellular responses. Cell morphology, spheroid diameter, cell concentration, viability, proliferation, and cytotoxicity were monitored 24- and 72- hpi. In a 2D cell culture, PBM at 5 J/cm 2 showed a significant increase in cell viability and proliferation. Cellular cytotoxicity was higher in 2D. Spheroids maintained steady size, increased viability, and proliferation peaking at 24 hpi but remaining stable over time at 10 J/cm 2. 2D cultures show an over expression of PBM-induced effects, while spheroids provide a more physiological and balanced response profile particularly at 10 J/cm 2.