The paper presents an HPLC method for cefazolin determination in human serum. The preparation step was based on serum protein precipitation with acetonitrile followed by supernatant evaporation and sample reconstitution in water before injection. The separation of cefazolin and internal standard cefamandole was performed at ambient temperature under isocratic conditions on LiChrosorb RP8-5 column (250mm×4.6mm) using the mixture: CH(3)CN:H(2)O:0.5M KH(2)PO(4) (100:894:6, v/v) as a mobile phase with a flow rate of 1.5mL/min. UV detection was performed at 272nm with LLOQ of 0.2μg/mL. The precision was satisfactory in the whole range tested with RSD of 2.3-12.5% (accuracy: from -2.3% to +3.6%) and of 1.7-7.1% (accuracy: from -3.5% to +1.1%) for intra- and inter-assay, respectively. The method stability was confirmed in a series of experiments including: freeze-thaw and short- and long-term stability testing. Finally, the procedure described was found resistant to potential human errors.