Abstract: Reversed-phase high performance liquid chromatography (RP-HPLC) was applied to the determination of 4 phenylketonuric characteristic acids, including 4-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, phenylacetic acid and phenylpyruvic acid in newborn urine. The urine sample (5.00 mL) was shaked with acetonitrile (5.0 mL) by swirling for 1min and centrifuged for 10 min. 4 analytes in the supernatant were separated by LC using Waters Symmetry C₁₈ column as stationary phase, and mixtures of acetonitrile and 0.15% (φ) phosphoric acid solution in various ratios as mobile phase in gradient elution. Detections were taken at the wavelengths of 210, 289 nm. Under the above conditions, the 4 characteristic acids were separated completely, and no interference by the endogenous substances in the urine was observed. Linearity for the 4 characteristic acids were kept between definite mass concentration ranges. Values of detection limits (3S/N) for the 4 characteristic acids were 0.43, 0.22, 0.62, 0.42 mg·L^-1 respectively. Tests for recovery were made by standard addition method at 3 concentration levels, giving results in the range of 89.1%-108%, with RSDs (n=6) less than 4%.