Introduction: Bupivacaine is a potent local anesthetic with side effects such as muscle twitching and irregular heartbeats. In the research, a sensitive and reliable extraction method based on the combination of the two-phase hollow fiber liquid-phase microextraction (HF-LPME) and response surface modeling is presented for preconcentration of bupivacaine prior to its determination by gas chromatography-mass spectrometry (GC-MS). Methodsand Materials:HF-LPME of bupivacaine was conducted using the polypropylene hollow fiber pieces (length of 8.8 cm, I.D=600 μm, thickness of 200 μm, and pore size of 0.2 μm) filled by 1-octanol as the acceptor phase, and a gas chromatograph (Agilent 7890A) coupled with a mass selective 5975C detector (GC-MS, Santa Clara, California, USA) was employed for analyte determination. To optimize the process, a Box-Behnken design with the variables of the extraction time (30, 60, and 90 min), pH (7, 9, and 11), and ionic strength (1, 2, and 3 mol L-1) was utilized. Results: The chromatographic peak areas of bupivacaine as the response were satisfactorily fitted to a quadratic response surface model (R2=0.9658 and F=21.98) which predicted the optimum conditions as the extraction time of 88.9 min, pH=11, and ionic strength of 2.1 mol L- 1. Based on the results, all the main factors and pH-extraction time interaction significantly affected the response. The calibration curve was linear in the range of 1-1000 µg L-1 (R2=0.9980), and detection and quantitation limits were obtained as 0.4 and 1.8 µg L-1, respectively. The method resulted in the high-efficiency range of 97.9%-100.0% for HF-LPME of bupivacaine in the actual samples. Conclusion: The results of this study suggested the proposed method as an efficient and environmentally friendly analytical method, just consuming 25 µL of the organic solvent for extraction of bupivacaine in the biological fluids.