Polygonum multiflorum Thunb, He-Shou-Wu in Chinese, is a widely used traditional Chinese medicine as a tonic and anti-aging agent. Since its major application form is water decoction, it was aimed to develop a simple and fast analytical method to analyze its hydrophilic bioactive components in its water extract. Therefore, pressurized liquid extraction and short-end injection micellar electrokinetic chromatography (MEKC) method were established for simultaneous determination of hydrophilic components including one alkaloid (hypaphorine), one stilbene (2,3,5,4’-tetrahydroxystilbene 2-O-β-D-glucoside), and five polyphenols (proanthocyanidin B1, proanthocyanidin B2, catechin, epicatechin, and gallic acid) in water extract of He-Shou-Wu. Method development was systematically investigated, the influence of parameters, such as pH, capillary temperature, separation voltage, concentration of phosphate, SDS and HP-β-CD, on the analysis were carefully investigated. Optimum separation was obtained within 14 min by using 50 mM phosphate buffer containing 90 mM SDS and 2% (m/v) HP-β-CD (pH 2.5) at 15 kV and 20 oC. All calibration curves showed good linearity (R2>0.9978) within test ranges. The overall LOD and LOQ were lower than 2.0 μg/mL and 5.5 μg/mL, respectively. The RSDs for intra- and inter-day of seven analytes V were less than 3.2% and 4.6%, and the recoveries were 97.0-104.2%. The validated method was successfully applied to the analysis of He-Shou-Wu samples collected from different regions of China, which is helpful for the quality control.