The aim of the present work is to develop and validate a simple, efficient, economical RP-HPLC method for the analysis of Sumatriptan (SMT) in samples obtained from in vitro transdermal permeation studies. A Phenomenex C18 reverse phase column (150 x 4.6mm, 5μm) with mobile phase containing 10mM ammonium acetate: methanol (75:25% v/v) at a flow rate of 1.2mL/min was used in isocratic mode and eluents were monitored at 230nm. The retention time of SMT was 3.61 min and showed a good linearity in the concentration range of 0.2-3μg/mL with a correlation coefficient >0.999. In vitro skin permeation studies were carried out using a Franz diffusion Cells. Absence of interference peaks at the retention time of SMT indicates that the method was specific for the analysis of SMT in samples obtained from in vitro transdermal permeation studies. The percent recoveries were ranged in between 98-102 (RSD < 2). The validation parameters like specificity, linearity, accuracy and limit of detection, limit of quantification, precision, robustness fulfilled regulatory requirements. The developed HPLC method was successfully used for the analysis of SMT in samples obtained from in vitro transdermal permeation studies.