Determination of pregabalin in urine samples was carried out by nonaqueous CE with TOF-MS via ESI, with a mixture of 10 mM ammonium formate and 0.05% acetic acid in methanol. By using TOF-MS, accurate mass information was obtained, thus causing a great improvement in qualitative ability. In order to avoid ionic suppression, urine samples dilution 1:10 was used. This was the only treatment to urine samples before the injection. Despite this dilution, the detection limit was as low as 0.03 g/mL for pregabalin. The method was validated with respect to accuracy, precision, and linearity, LOD, and LOQ. This method was applied to the analysis of urine samples from seven different cancer patients undergoing treatment with pregabalin. The developed method may find wide application for the routine determination of pregabalin in biological samples in order to establish a more efficient and safe dosage.