The new method for HPLC determination of piracetam in human plasma was developed and validated by a new approach. The simple determination by UV detection was performed on supernatant, obtained from plasma, after proteins precipitation with perchloric acid. The chromatographic separation of piracetam under a gradient elution was achieved at room temperature with a RP-18 LiChroSpher 100 column and aqueous mobile phase containing acetonitrile and methanol. The quantitative determination of piracetam was performed at 200 nm with a lower limit of quantification LLQ=2 μg/ml. For this limit, the calculated values of the coefficient of variation and difference between mean and the nominal concentration are CV%=9.7 and bias%=0.9 for the intra-day assay, and CV%=19.1 and bias%=−7.45 for the between-days assay. For precision, the range was CV%=1.8÷11.6 in the intra-day and between-days assay, and for accuracy, the range was bias%=2.3÷14.9 in the intra-day and between-days assay. In addition, the stability of piracetam in different conditions was verified. Piracetam proved to be stable in plasma during 4 weeks at −20 °C and for 36 h at 20 °C in the supernatant after protein precipitation. The new proposed method was used for a bioequivalence study of two medicines containing 800 mg piracetam.