A reversed-phase high-performance liquid chromatography method was established for the simultaneous determination of four major constituents, namely myricitrin, hyperin, quercitroside, and quercetin in Folium Rhododendri Micranthi. The optimal conditions of separation and detection were achieved on a C18 analytical column with a gradient mobile phase consisting of acetonitrile and 1% acetic acid at the flow rate of 1.0 mL/min, and detection wavelength was set at 355 nm. All calibration curves showed good linear regression (r > 0.9993) within test ranges. The reproducibility of relative standard deviations was less than 2.3%, and recovery of analytes was greater than 99.4%, respectively. The method was successfully applied to determine the contents of four compounds in Folium Rhododendri Micranthi. The results indicated that the contents of myricitrin, hyperin, quercitroside and quercetin in Folium Rhododendri Micranthi were 0.166%, 0.303%, 0.299%, and 0.053%, respectively.