A rapid,sensitive,selective and validated reverse phase high-performance liquid chromatography(RP-HPLC)method for the estimation of paclitaxel in micro-sample of rat plasma and in culture of cancer cells was performed in this study.The mobile phase consisted of an optimized mixture of methanol:water:trifluroacetic acid(80:20:0.1,v/v/v).Column elution at a flow rate of 1 mL/minute with UV detection at 225 nm at room temperature was used.The RP-HPLC method was successfully applied for the determination of paclitaxel in plasma samples and in culture of cancer cells with nano-quantity of estimation.The validation studies were performed in accordance with the International Conference on Harmonization(ICH)guidelines.The intra-and inter-day precision showed that the coefficients of variation ranged from 1.07%to 4.27%at different levels of concentrations.To the best of our knowledge,this study also reported for the first time the optimization of different solvents for effective extraction of paclitaxel wherein tert.-butyl methyl ether(TBME):diethyl ether(DEE)in 50:50 v/v composition was found most efficient with extraction efficiency ranging between 77.99%and 91.74%and between 76.14 and 93.66%in the plasma and cell culture,respectively.This proposed method was successfully applied to study the pharmacokinetics of paclitaxel and the influence of verapamil and all-trans retinoic acid(atRA)on paclitaxel pharmacokinetics in rat models.This proposed method might emerge as a valuable aid in the laboratory monitoring of paclitaxel in a variety of in vitro as well as in vivo scenarios. A rapid, sensitive, selective and validated reverse phase high-performance liquid chromatography (RP-HPLC) method for the estimation of paclitaxel in micro-sample of rat plasma and in culture of cancer cells was performed in this study. Mobile phase consisted of an optimized mixture of methanol: water: trifluroacetic acid (80:20: 0.1, v / v / v) .Column elution at a flow rate of 1 mL / minute with UV detection at 225 nm at room temperature was used.The RP- HPLC method was successfully applied for the determination of paclitaxel in plasma samples and in culture of cancer cells with nano-quantity of estimation. Validation experiments were performed in accordance with the International Conference on Harmonization (ICH) guidelines. Intra-and inter- day precision showed that the coefficients of variation ranged from 1.07% to 4.27% at different levels of concentrations. To the best of our knowledge, this study also reported for the first time the optimization of different solvents for effective extraction of p acitaxel for tert.-butyl methyl ether (TBME): diethyl ether (DEE) in 50:50 v / v composition was found most efficient with extraction efficiency ranging between 77.99% and 91.74% and between 76.14 and 93.66% in the plasma and cell culture, respectively. This proposed method was successfully applied to study the pharmacokinetics of paclitaxel and the influence of verapamil and all-trans retinoic acid (atRA) on paclitaxel pharmacokinetics in rat models. This proposed method might emerge as a valuable aid in the laboratory monitoring of paclitaxel in a variety of in vitro as well as in vivo scenarios.