In this paper, a quantification method of the carbohydrate is proposed for samples of spot tests using the thin-layer chromatography (TLC) based on the scanned image analysis compensating lack of overlap between each spot. The color density of image data is modeled by 2-dimensional Gaussian function. Parameters in the Gaussian function are estimated by calculating marginal distribution and using least squares (LS) method. Sample image of the spots on the TLC plate are quantified as calculating the volume of Gaussian function. A numerical example is then carried out for quantifying the glucose which is an important and commonly unit of the carbohydrate. The efficiency of our method is pointed out comparing with two non-compensated methods. In addition, in order to investigate the performance of proposed method, calibrated values are also compared with results using the high performance liquid chromatography (HPLC).