Ezetimibe is the first in a new class of antihypercholesterolemic drugs. Since it has not long been available on the market, many of its properties may still be revealed. Analytical methods for its determination are scarce, especially regarding serum samples. A simple, fast, and effective high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the determination of ezetimibe concentration in human serum has therefore been developed. Three mobile phases were analysed, and original modifications to the concentration and flow parameters were made. Of five potential internal standards (IS), only nitrendipine was found to be suitable. The analytical wavelength was chosen based on the absorption spectrum of ezetimibe in the mobile phase. Finally, an extraction analysis was performed using two different solvents, and the extrahent volume was optimised. The final method developed was as follows. Single extraction of 1 mL serum sample, spiked with IS, was performed using 10 mL of methyl-t-butyl ether. Separation was obtained at ambient temperature on a Waters C18 Symmetry Shield (4.6 mm × 250 mm, 5 μm) column. The isocratic mobile phase was composed of acetonitrile and 0.1 M ammonium acetate aqueous solution 55:45 (v/v), set at flow rate of 0.75 mL min−1. Ezetimibe was detected at a wavelength of 232 nm after 5.49 min, and the IS was detected at 8.05 min. The developed method has been validated according to ICH standards. It was found to be specific, precise, accurate and linear over the range 10–800 ng mL−1 with R2 > 0.998, and detection and quantification limits of 4.60 ng mL−1 and 13.94 ng mL−1, respectively. The method has been applied to clinical serum samples. The developed technique allowed for successful in vivo assessment of ezetimibe concentrations in samples obtained from hypercholesterolemia patients who are chronically receiving the drug.