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A simple and rapid HPLC method is developed for the determination of two serotonin-norepinephrine-reuptake inhibitors (duloxetine and venlaflaxine) and two selective serotonin-reuptake inhibitors (fluoxetine and paroxetine) in human biofluids. Separation was performed on an Inertsil ODS-3 column (250 x 4.0 mm, 5 pm) with acetonitrile ammonium acetate (0.05 M, 41:59 v/v) at 235 nm, within 7 min. SPE on Oasis (R) HLB cartridges was applied for the isolation of analytes from biofluids. The developed methodology was validated in terms of sensitivity, linearity, accuracy, precision, stability and selectivity. Relative standard deviation was less than 10.4%. Limit of detection was 0.2-0.6 ng/mu l in blood plasma and 0.1-0.8 ng/mu l in urine. The method was successfully applied to biofluids from a patient under duloxetine treatment.