AIM: To establish reverse phase high performance liquid chromatography (RP-HPLC) method to determine the concentration of ropivacaine and bupivacaine in maternal and fetus plasma. METHODS: Agilent HPLC instrument was used with the column: Dikma-C 18(5 μm,4.6×150 mm). Internal standard was rosiglitazone. The mobile phase was composed of NaH 2PO 4(10 mmol·L -1,pH 3.0)∶CH 3CN(78∶22,V/V).Flow rate was 1.0 mL·min -1.Detection wavelength was 210 nm. RESULTS: The linear coefficient relation of ropivacacine and bupivacaine were Y=0.0295X- 0.0298 and Y=0.0287X+0.0271(r=0.9998,n=7). The minimum detection limit of ropivacaine and bupivacaine were both 0.01 mg·L -1. The linear ranges were both (0.01-5.0) mg·L -1. The average recovery rates of method were 99.82% and 101.01%. The coefficient variations of intra- and inter-day of ropivacaine for 0.1, 1, 5 mg·L -1 were less than 2.47% and 3.75%. The coefficient variations of intra- and inter-day of bupivacaine for 0.1, 1, 5 mg·L -1 were less than 2.69% and 4.75%. The concentration of sixty samples of ropivacaine was 0.15-0.7 mg·L -1, and that of bupivacaine was 0.1-0.58 mg·L -1. CONCLUSION: The RP-HPLC method is simple, sensitive and accurate. It is applicable to determine the concentration of ropivacaine and bupivacaine.