A highly sensitive precolumn high performance liquid chromatographic（HPLC） method for the simultaneous determination of free hydroxyproline（Hyp） and proline（Pro） in human urine was developed.Effects of reaction temperature,pH value,time,concentration of labeling reagent,composition of moblie phase,concentration and pH value of buffer,and column temperature were investigated in detail.Under the optimal conditions,the calibration curves showed good linear relationship in the concentration ranges of 5-100 μmol/L for hydroxyproline and 5-250 μmol/L for proline.The correlation coefficients were 0.999 4 for hydroxyproline and 0.999 5 for proline.The detection limits（S/N=3） for hydroxyproline and proline were 0.50 nmol/L and 0.20 nmol/L,respectively.The recoveries（n=5） for hydroxyproline and proline were in the ranges of 95%-99% and 96%-102% with RSDs of 2.1%-4.3%and 2.0%-4.8%,respectively.This method can be used for the qualitative analysis of free hydroxyproline and proline in human urine sample.