Objective To establish a quality control method for simultaneous determination of multiple components in gamboge. Methods A single reference standard for the determination of multiple components(SSDMC) with HPLC was proposed. Seven major components of gamboge including gambogenic acid(S), β-morellic acid(C1), 2R-30-hydroxygambogic acid(C2), isogambogenic acid(C3), gambogellic acid(C4), 2R-gambogic acid(C5), and 2S-gambogic acid(C6) were simultaneously analyzed using gambogenic acid as reference standard. The credibility and feasibility of SSDMC method were validated with respect to linearity, limits of detection and quantification, precision, stability, repeatability, accuracy, ruggedness, and robustness. The relative conversion factors(RCFs) of S and C1-6 were calculated. Twelve batches of gamboge including crude and processed products were successfully analyzed by applying the SSDMC and traditional external standard(ES) methods. Results The SSDMC method was credible and feasible. The RCFs of S and C1-6 were 1.000, 0.913, 0.864, 1.064, 0.777, 0.921, and 0.919, respectively. No significant difference was observed in the contents of the seven components between SSDMC and ES methods. The heat-processing technique caused a reduction in the seven components. Conclusion SSDMC is a simple, reliable, and effective method for the analysis of the complex multiple components in gamboge, and it is also a practical and economical approach. Methods A single reference standard for the determination of multiple components (SSDMC) with HPLC was proposed. Seven major components of gamboge including gambogenic acid (S), β-morellic (C3), gambogellic acid (C4), 2R-gambogic acid (C5), and 2S-gambogic acid (C6) were the same as used in the analysis of gambogenic acid as reference standard. The credibility and feasibility of SSDMC method were validated with respect to linearity, limits of detection and quantification, precision, stability, repeatability, accuracy, ruggedness, and robustness. The relative conversion factors (RCFs) of S and C1-6 were calculated. Twelve batches of gamboge including crude and processed products were successfully analyzed by applying the SSDMC and traditional external standard (ES) methods. Results The SSDMC method was credible and feasible. Th e RCFs of S and C1-6 were 1.000, 0.913, 0.864, 1.064, 0.777, 0.921, and 0.919, respectively. Significant difference was observed in the contents of the seven components between SSDMC and ES methods. The heat-processing technique caused a reduction in the seven components. Conclusion SSDMC is a simple, reliable, and effective method for the analysis of the complex multiple components in gamboge, and it is also a practical and economical approach.