A sensitive and rapid HPLC assay for the determination of fluconazole in serum is described. HPLC-integrated sample preparation allows direct injection of serum samples without any pretreatment. The in-line extraction technique is carried out by automatically switching from the extraction column (Lichrospher® ADS C ₈) to the analytic column (Nucleosil C ₁₈). After 6 minutes the matrix passes the extraction column, and the retained analyte is quantitatively transferred to the analytic column, where separation by isocratic HPLC is performed. The extraction eluent is sodium dihydrogen phosphate buffer, pH 5.0 (50 mM), and the analytic eluent is acetonitrile/sodium dihydrogen phosphate buffer, pH 5.0 (50 mM) (26.8/73.2, vol/vol). Fluconazole is detected according to its absorption maximum at 210 nm. The lower limit of quantification (LLOQ) is 0.65 μg/mL, the limit of detection (LOD) is 0.2 μg/mL, and the quantification range is 0.65-23.3 μg/mL. The assay was precise with a between-run coefficient of variation of ≤ 5.59%. The within-run accuracy was 99.8% and 103.4%, and the between-run accuracy was 99.2% and 99.7%, respectively, for the concentrations 23.3 μg/mL and 1.3 μg/mL. The recovery was 78%. The described procedure allows sample cleanup and determination within 20 minutes, thereby facilitating drug monitoring in clinical routine. The method was applied successfully.