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UHPLC–MS/MS method for vancomycin determination in human serum, urine and peritoneal/pleural effusion was developed and fully validated.A simple and rapid sample pretreatment using small volume of biological liquids is suitable for large sample series.The method uses unified conditions for all liquids and would be applicable to the study of the influence of liquid sequestration in the “third space” of patients undergoing vancomycin therapy.Management of the therapy of life-threatening bacterial infection is extremely based on an optimal antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur.A UHPLC–MS/MS method operating in electrospray (ESI) positive ion mode was applied for the determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was composed of dilution and simple protein precipitation where only a small volume (50 μL) of serum, urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a Meteoric Core C18 BIO column (100 × 4.6 mm, 2.7 μm) by gradient elution with 0.1% formic acid in water and acetonitrile. The total time of analysis was 4.5 min. The method was found to be linear in the range of 2–60 μM (or 0.5–10 μM) for serum, 0.27–10 μM (or 2–60 μM) for peritoneal/pleural effusion and 25–300 μM for urine, which was adequate for the determination of vancomycin in patient samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%.The UHPLC/MS–MS method offers a fast and reliable approach to determine vancomycin concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural effusion) with a simple sample pretreatment that was the same for all selected specimens. This method should be applicable to large sample series in clinical (pharmacokinetic/pharmacodynamic) studies.