Objective:To investigate the effects of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy on apoptosis in cervical cancer HeLa cells.Methods:The ultraviolet absorption spectrum and fluorescence spectrum of protoporphyrin Ⅸ were measured using a UV spectrophotometer and a fluorescence spectrometer, respectively. The cell viability of HeLa cells treated with different concentrations (0, 10, 20, 40, 60, 80, 100, 120 and 140 μg/ml) of 5-ALA was assessed using the CCK-8 assay. Hoechst 33342 and dichlorofluorescein diacetate (DCFH-DA) staining methods were utilized, and the production of protoporphyrin Ⅸ and reactive oxygen species in the control group and laser + 150, 200 μg/ml 5-ALA groups were observed using a laser scanning confocal microscope. Apoptosis in the control group, laser group, 5-ALA group, and laser + 5-ALA group was detected using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining and live-dead cell staining methods.Results:The UV absorption spectrum showed an absorption peak of protoporphyrin Ⅸ at 406 nm, and the fluorescence spectrum revealed a distinct characteristic peak at 635 nm. The CCK-8 assay result indicated a gradual decrease in cell viability with increasing concentrations of 5-ALA. Laser scanning confocal microscopy results demonstrated that 5-ALA could be converted into protoporphyrin Ⅸ within cells, emitting red fluorescence. In the laser + 5-ALA group, the green fluorescence of reactive oxygen species from HeLa cells labeled with DCFH-DA fluorescent probes were detected. Flow cytometry results showed that the apoptosis rates in the control, laser, 5-ALA, and laser + 5-ALA groups were 12.55%, 12.41%, 13.51%, and 28.31%, respectively. Live-dead cell staining indicated a significant occurrence of apoptosis in laser + 5-ALA group.Conclusions:5-ALA mediated photodynamic therapy can induce apoptosis in HeLa cells through the generation of reactive oxygen species.