Abstract: Objective In order to guide clinical differential and diagnosis of OSCC, the florescence intensities were detected by the microplate reader to test the endogenous protoporphyrin IX which was produced by 5-ALA in varying degrees of differentiation oral squamous cell carcinoma. Methods 2×104 of SAS cells were cultivated in 96-well plates, the fluorescence intensities of different concentrations of PpIX aquenous solution were detected to make the standard curve of PpIX. 5-ALA of 0, 0.5, 1, 1.5, 2mmol/L were added into SAS, HSC-3 and HSC-4 respectively, and then the fluorescence intensities were detected at 0, 2, 4, 6, 8, 10 hours. Results The fluorescence intensities which were detected by microplate reader were linearly related with PpIX content. The best metabolite concentration of 5-ALA was 1 mmol/L in OSCC cells, and the metabolic time was 4 hours. The fluorescence intensity of 5-ALA in the differentiating SAS cells was significantly higher than the differentiated HSC-3 and HSC-4 cells (P< 0.05）. Conclusion 5-ALA produces different fluorescence intensity in varying degrees of differentiation OSCC cells. These results can be used in the differential diagnosis of clinical OSCC.