A simple and rapid high-performance liquid chromatography method coupled with UV detector was developed and validated for the simultaneous determination of ropivacaine, bupivacaine and dexamethasone in biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres within 11 min. Chromatographic separation was performed on a XDB-C(18) column using a mobile phase comprised of acetonitrile-NaH(2)PO(4) buffer (pH 3.5, 30 mM) (30:70, v/v) with a flow rate gradient program. The method was in good linearity (r>0.999) over the range of 0.025-40.0 microg/ml for ropivacaine and bupivacaine, and 0.05-40 microg/ml for dexamethasone. The method was proved to be precise with intra- and inter-day precision less than 3.0% and 6.0% for all drugs and accurate with intra- and inter-day accuracy between -8.0% to 4.5% and between -5.0% to 5.5% for all drugs. The assay was rapid, simple and easy to apply. Therefore, it was very suitable for routine determination and quality control of ropivacaine, bupivacaine and dexamethasone in PLGA microspheres.