A novel bio-analytical method was developed and validated for the quantitative determination of erlotinib in rat serum by using the liquid-liquid extraction extraction chromatography and tandem mass spectrometric detection (HPLC-MS/MS). Separation of erlotinib from the endogenous substances is achieved after liquid-liquid extraction by using HPLC-MS/MS system. Erlotinib was eluted in isocratic mode with acetonitrile and 0.1% formic acid in 5 mM ammonium formate ( 80:20, v:v) at a flow-rate of 0.3 mL/min on phenominex, Gemini C18, 50*2.0 mm, 5μm particle size column. Imipramine was used as the internal standard. The liquid-liquid extraction recovery was found 76% indicates good recovery. The present method was found to be sensitive and selective at very low levels of linearity range 1-1000ng /mL based on a sample volume of 50 μL, with a linear correlation coefficient of ≥ 0.99. The validation results demonstrated that the present method was found to be precise and accurate. The stability tests indicated that the erlotinib in rat serum is stable for three freeze-thaw cycles at both -20 ºC and -70 ºC, 18-h ambient storage, 15-day frozen storage at both -20 ºC and -70 ºC. The results also showed no significant matrix effect (<4.2%). The present method was found to be sensitive and selective at very low levels of linearity range 1-1000ng. The validated method has been successfully applied to support a preclinical pharmacokinetic study.