Dioscorea hispida Dennst. (Dioscoreaceae) is a valuable source of edible starch. Its dried tuber has been used as a crude drug in Thai remedy named Thoraneesanthakhat. It has traditionally been used to treat constipation. D. hispida contains neurotoxic dioscorine, an isoquinuclidine alkaloid causes nervous system paralyze. Even though there are traditional wisdoms to detoxify the tuber, emphasis on quality control of dioscorine content is essential for D. hispida development. This research was to establish dioscorine content of D. hispida dried tubers in Thailand by TLC - densitometry and image analysis. Dried tubers collected from 14 areas throughout Thailand were successively extracted in ethanol by soxhlet apparatus and analyzed for dioscorine by TLC. Aluminium oxide 60 GF254 neutral was used as stationary phase and methanol-chloroform (3:97) was used as mobile phase. Intensity of dioscorine under UV254 was analyzed using Camag TLC scanner 3 and Scion image software. Each sample was quantitated in triplicate. The grand average dioscorine content of the dried tuber was 0.72 ± 0.07 % w/w (mean ± 3 SD = 0.51 - 0.93 % w/w) by TLC-densitometry and 0.66 ± 0.07 % w/w (mean ± 3 SD = 0.44 - 0.88 % w/w) by TLC image analysis. Method validation showed that TLC image analysis could be altered to high cost densitometer for dioscorine quantitation. TLC image analysis for dioscorine quantitation was developed and dioscorine content in D. hispida dried tuber was established. This parameter could be used as a specification for quality control of D. hispida dried tuber in Thailand.