A chiral UPLC-MS/MS method was developed and validated to determine oxiracetam enantiomers in human plasma, urine, and feces. The R-Oxiracetam and S-Oxiracetam were quantified using a CHIRALPAK ®AD3 column at 25 ℃, and the resolution was greater than 3.2. The S-Oxiracetam is the eutomer that isresponsible for the treatment of various brain damage. Isocratic elution was conducted at a flow rate of 0.9 mL/min for 6 min using the mixture of methanol and acetonitrile (methanol:acetonitrile, 15:85) containing 0.3‰ formic acid. The methods showed linearity at the range of 0.5-100 µg/mL for each oxiracetam enantiomer. A comprehensive validation process was carried out, covering aspects including linearity, selectivity, carryover, accuracy, precision, interferences, matrix effect, recovery, dilution integrity and stability in matrix and solution. The validated methods were successfully applied to quantifying R-Oxiracetam and S-Oxiracetam in human plasma, urine, and feces of 12 healthy subjects treated with either a single dose of 2 g S-Oxiracetam injection or 4 g Oxiracetam injection in a phase-I clinical trial. There was no significant difference for plasma pharmacokinetic parameters of S-Oxiracetam between the two regimens (P>0.05). The S-Oxiracetam and Oxiracetam were primarily eliminated through urine in their original form, with cumulative excretion rates of 92.16% and 85.92%, respectively, within 24 h after administration. Enantiomers interconversion was not observed in the plasma, urine, or feces. The results of this study suggest that replacing 4 g Oxiracetam injection with 2 g S-Oxiracetam injection could offer clinical benefits by lowering the dosage and mitigating potential risks, based on the pharmacokinetic characteristics.