Liquid phase microextraction based on hollow fiber is one of the liquid phase microextraction techniques that have been considered in recent years. In three phase hollow fiber, an aqueous acceptor phase is used for the extraction of analytes from sample solution that can be injected only to high performance liquid chromatography and capillary electrophoresis. So, for extraction of compounds before analysis with gas chromatography, a two phase hollow fiber is used. In this study, instead of two phase, a three phase HF-LPME was used for the extraction and preconcentration of desipramine before analysis with gas chromatography. Methanol was used as an acceptor phase, instead of aqueous phase. So, this new three phase method can be used for the extraction of analytes before inject to gas chromatography. Determination of desipramine in biological samples such as plasma and urine was performed using in-syringe derivatization and followed by gas chromatography-nitrogen phosphorus detector. In the hollow fiber liquid-liquid-liquid microextraction, the lumen of hollow fiber was filled with methanol as an acceptor phase. Then a layer of organic phase (n-dodecane) was impregnated into the pores of an 8 mm porous hollow fiber. The fiber was then immersed into 3 ml of a basic aqueous sample. Due to its low cost, the hollow fiber was disposed after a single extraction that eliminated the possibility of carry over effects. After extraction, desipramine was derivatized using acetic anhydride as derivatization reagent. Then, the derivatized analyte was injected to the GC-NPD. Derivatization reaction was performed using 0.6 µL of acetic anhydride at room temperature for 2 min. The effect of parameters influencing the extraction efficiency such as organic solvent type, concentration of sodium hydroxide in donor solution, sample stirring rate, salt addition, extraction temperature and extraction time were studied. The best extraction efficiency was obtained using 0.05 M sodium hydroxide as donor phase, stirring rate of 900 rpm, 25 min extraction time, no salt addition, at room temperature. The intra-day and inter-day relative standard deviation of the method were 2.6% and 7.7 %, respectively. The enrichment factor, limit of detection and correlation coefficients were 90, 0.02 ?g/L and 0.9986, respectively.