In the present study, a simple and rapid reversed-phase HPLC procedure has been developed for the simultaneous determination of eight fat-soluble vitamins (retinol, menadione, menaquinone, delta-tocopherol, cholecalciferol, alpha-tocopherol, alpha-tocopherol acetate and phylloquinone) in biological fluids: blood serum and urine. The analytical column, Phenomenex Luna C18 (150 mm x 4.6 mm) 3 microm, was operating at ambient temperature. Mobile phase consisted of a mixture of CH3OH-CH3CN delivered using a linear gradient, starting with a composition of 50-50% v/v and ending at 30-70% at a flow rate of 1.3 ml/min. Xanthophyll was used as internal standard (2 ng/microl). Detection and identification was performed using a photodiode array detector. Eluent monitoring was achieved at 280 nm for vitamins and 450 nm for the internal standard. However, quantitation was performed at maximum wavelength for each vitamin. Detection limits were found in the range of 1.4-6.6 ng per 20-microl injected samples, while linearity held up to 25 ng/microl. The statistical evaluation of the method was examined performing intra-day (n = 6) and inter-day calibration (n = 7) and was found to be satisfactory, with high accuracy and precision results. The biological fluids were treated using solid-phase extraction cartridges, to remove all endogenous interferences from sample matrix. The solid-phase extraction protocol was optimized in terms of retention and elution. High extraction recoveries from biological matrices: blood serum and urine, (average recovery ranging between 95 and 97.6% for blood serum and between 94.2 and 95.8% for urine) were achieved for the eight fat-soluble vitamins, using Cyclohexyl J.T. Baker SPE cartridges with methanol as eluent, requiring small volumes, 100 microl of blood serum and 100 microl of urine.