trans-4-Hydroxy-L-proline is one of the valuable chiral building block in the production of many pharmaceuticals.A quantification method of trans-4-hydroxy-L-proline and L-proline from microbial transformation by using reverse phase high performance liquid chromatography （RP-HPLC) was developed.9-Fluorenylmethyl chloroformate（FMOC-Cl) was used as pre-column derivation agent,and the derivatization was proceeded in sodium tetraborate buffer solution（pH 9.9) containing acetonitrile-water（66∶34).The solution then was separated on an Agilent Extend C-18 column by using 0.1% trifluoroacetic acid and acetonitrile as mobile phase,and detected with UV detector at 263 nm.The linear ranges of two amino acid were in the range of 0.01-5.00 mg/mL,with correlation coefficients greater than 0.999.The limits of detection（LOD) were in the range of 5.00-7.00 ng/L,and the average recoveries of trans-4-hydroxy-L-proline and L-proline were 98.9%-102% and 97.9%-100%,respectively.This method provides an effective way to quantify L-proline and trans-4-hydroxy-L-proline in microorganism fermented liquid system with good reproducibility,and high precision.