Objective: The objective of this study was to obtain an optimum and valid method of analysis to determine the levels of disodium 5'-guanylate (DSG) and disodium 5'-inosinate (DSI) in six samples of spices. Methods:The optimum method was obtained using silica gel 60 F 254 as the stationary phase and isopropanol:water:25% ammonia at a ratio of 6:3:1 (v/v) as the mobile phase. The developed spots were scanned using a densitometer in absorbance mode at 260 nm. The methods were valid based on the accuracy criteria (DSG, 99.11-99.96%, and DSI, 98.56-101.05%), precision (DSG, 1.09%, and DSI, 0.49%), and linearity (DSG, r=0.9909, and DSI, r=0.9976). Results:The results showed that the levels of DSG in samples A, B, C, D, E, and F were 0.70%, 0.79%, 0.78%, 0.99%, 1.08%, and 1.08% and those of DSI were 0.66%, 0.74%, 0.71%, 0.66%, 0.54%, and 0.67%, respectively. Conclusion:The optimum conditions of DSG and DSI for thin-layer chromatography-densitometry were obtained with silica gel 60 F 254 as the stationary phase, isopropanol:water:25% ammonia (6:3:1) as the mobile phase, and a maximum wavelength of about 260 nm. Validation results indicated that the accuracy of the analytical method for DSG was about 99.11-99.96% with a coefficient variation (precision) of 0.70-1.41%, while that for DSI was 98.56-101.05% with a coefficient variation of 0.23-0.75%. The correlation coefficients for the analytical method for DSG and DSI were 0.9909 and 0.9976, respectively. The results determined that the levels of DSG and DSI in samples A, B, C, D, E, and F were 0.70%/0.60%, 0.79%/0.74%, 0.78%/0.71%, 0.99%/0.66%; 1.08%/0.54%, and 1.08%/0.67%, respectively.