Hollow fibre liquid-phase microextraction (LPME) and desorption electrospray ionization mass spectrometry (DESI-MS) were evaluated for the identification and quantification of basic drugs in human urine samples. The selective extraction capabilities of three-phase LPME provided a significant reduction in the matrix effects otherwise observed in direct DESI-MS analysis of urine samples. Aqueous LPME extracts (in 10 mM HCl) were deposited on porous Teflon, dried at room temperature, and the dried spots were then analyzed directly with DESI-MS in full scan mode. Pethidine, diphenhydramine, nortriptyline, and methadone were used as model compounds for identification, and their limits of identification were determined to be 100, 25, 100, and 30 ng/mL, respectively. In a reliability test with 19 spiked urine samples, 100 % of the positive samples containing the model drugs in concentrations at or above the limit of identification were identified. Diphenhydramine was used as a model compound for quantitative analysis with diphenhydramine-d5 as an internal standard. The calibration curve was linear in the range 50–2000 ng/mL (R2 = 0.992) with a limit of quantification at approximately 140 ng/mL. The intra- and inter-day relative standard deviations were <9.5 %. In a reliability test with six spiked urine samples, deviations between the measured and the true values for diphenhydramine were in the range 0.2–22.9 %. Copyright © 2011 John Wiley & Sons, Ltd.