Nonaqueous capillary electrophoresis (NACE) is very well suited for online coupling with mass spectrometry due to the relatively high volatility and low surface tension of most organic solvents. Here we present a quantitative NACE-ESI-MS/MS method for separating and determining physcion, chrysophanol, and aloe-emodin in rhubarb. Dantron was used as an internal standard to ensure accuracy and reproducibility in quantitative analyses. Parameters including the pH, background electrolyte (BGE) composition, flow-through microvial chemical modifier solution composition, and modifier solution flow rate were carefully optimized. The developed method was validated by assessing its precision, LODs, and linear range. The contents of physcion, chrysophanol, and aloe-emodin in rhubarb were determined to be 0.22%, 1.0%, and 0.17%, respectively.