The Ministry of Food and Drug Safety is amending its test methods for dietary supplements (functional health food) to establish regulatory standards and specifications in South Korea. In this study, we develop analytical test methods to improve the sensitivity and selectivity of the chitosan test method. We develop liquid chromatographic-tandem mass spectrometry for a chitosan analysis using acid hydrolysis to glucosamine. This method can be used to characterize and quantify dietary supplement formulations containing chitosan. The procedure was validated in the 78 ~ 2,500 ng/mL range, and standard calibration curves presented linearity with the coefficient of determination (r²) > 0.999. The limits of detection and quantitation were 51 ng/mL and 154 ng/mL, respectively. The recovery results ranged between 94.8 ~ 102.9% at three different concentration levels with relative standard deviations less than 3.1%, and precision (0.8 ~ 2.5%) was obtained. These are in accordance with the established validation criteria (Association of Official Analytical Chemists). Subsequently, our research provides scientific evidence for amending the chitosan test method for the Health Functional Food Codes.