Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.