Rhinacanthus nasutus (L.) Kurz, a plant widely used in Thai traditional medicine for treating skin ailments, contains rhinacanthin C as its primary antifungal compound, predominantly found in leaves and roots. Despite its integration into numerous health products, there is limited scientific data on the variability of rhinacanthin C levels across different raw material sources. This study aimed to quantify rhinacanthin C in R. nasutus raw materials using HPLC methodology. Analysis employed a C18 column with a mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in acetonitrile and 0.1% TFA in water (75:25 v/v) at 1 mL/min flow rate. Detection utilized a photodiode array detector (PDA) set to measure UV absorbance at 254 nm. Results from three sources indicated rhinacanthin C content ranged from 0.01% to 1.27% w/w in leaves and 1.11% to 2.42% w/w in roots. Evaluation of raw materials from four suppliers revealed levels below 0.05% w/w. These findings highlight significant variation in rhinacanthin C content among different sources, underscoring the importance of rigorous content analysis for quality control and selection of optimal raw material sources.