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This study aimed to develop and validate a HPLC based analytical methodology for simultaneous determination of acyclovir and curcumin within microparticles. Chromatographic separation was achieved by employing a Fenomenex C18 column as stationary phase and a ternary mixture of acetonitrile, 0.1% phosphoric acid, and methanol (50: 40: 10) as the mobile phase. The validated method proved to be linear in the range of 0.5-30 μg. mL− 1 and 0.5-20 μg. mL− 1 for acyclovir and curcumin, respectively. Detection and quantification limits for acyclovir were, respectively, 83.62 ng. mL− 1 and 109.52 ng. mL− 1, while for curcumin the values were 91.61 ng. mL− 1 and 128.71 ng. mL− 1, what assures the methodology sensitivity. The method was also precise (1.2% RSD for acyclovir and 1.38% RSD for curcumin), besides showing recovery rates close to 100% for both of two drugs when accuracy was accessed. Minor alterations over chromatographic setup have confirmed methodology robustness. The present methodology proved to be capable of detecting and quantifying acyclovir and curcumin at polymeric microparticles in a single run, showing itself as an analytical alternative to be employed in the quality control for this dosage form.