OBJECTIVE: To establish a method for the determination of related substances in Levo-cetirizine hydrochloride chewable tablet. METHODS: RP-HPLC was performed on the column of Venusil XBP-CN with mobile phase of acetonitrile-0.05 mol/L Sodium dihydrogen phosphate solution(pH value adjusted to 3.0 by phosphoric acid)(60 ∶ 40,V/V) at a flow rate of 1.0 ml/min, the detection wavelength was 230 nm, column temperature was 25 ℃, and volume injection was 20 μl. RESULTS: Levo-cetirizine hydrochloride chewable tablet showed good separation with related substances; the linear range of levo-cetirizine hydrochloride was 1.2-2.8 μg/ml (r=0.999 9); the quantification limit and detection limit was 1.4 ng/ml and 0.3 ng/ml, respectively; RSDs of precision, stability and reproducibility tests were lower than 1%; recovery was 96.3%-105.0% (RSD=1.7%,n=9); the contents of related substances were no more than 0.17%. CONCLUSIONS: The method is simple, sensitive, reproducible, accurate and reliable, and can be used for the determination of related substances in Levo-cetirizine hydrochloride chewable tablet.