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Capillary zone electrophoresis was employed to determine cefazolin, a first‐generation cephalosporin antibiotic, in plasma and microdialysis samples from patients. To shorten the analysis, the samples were injected from the short end of the capillary, resulting in a separation time of < 3 min. Due to a high ionic strength of the biological matrices it was necessary to optimize the stacking conditions. For microdialysis samples a 1:10 dilution with water before injection was sufficient to obtain good peak shape. For plasma samples a protein removal step was required to obtain clean electropherograms and a good peak shape. Acetonitrile was used as precipitant resulting in an enhanced sample stacking in comparison to water dilution. The disadvantage of using acetonitrile was severe evaporation loss making quantitation impossible. A self‐sealing film was used to seal each individual sample vial to suppress evaporation during long‐term sequences. The calibration curves for spiked plasma and cefazolin in Ringer's solutions were linear in the range from 2–500 and 2.5–100 μg/mL, respectively. Limits of detection were 1.0 and 2.0 μg/mL in plasma and microdialysis samples, respectively. The assay was successfully applied to plasma and microdialysis samples obtained in vivo from the interstial space fluid of subcutaneous adipose and muscle tissue of patients undergoing cardiac surgery.