Abstract: The main purpose of the present study was to develop and validate a high performance thin layer chromatographic (HPTLC) method for quantitative determination of an antiviral agent, zanamivir in pure drug and diskhaler powder formulation. Chromatography was performed on aluminum TLC plates pre-coated with silica gel 60F254, employing a mixture of chloroform:methanol:ammonia (9.5:3.2:0.2,v:v:v) as mobile phase. The TLC scanner was operated in the absorbance mode at a wavelength of 230 nm for evaluation of chromatograms. The system has given well resolved peak of zanamivir (Rf = 0.56). The linearity of the method was established in the range of 20-300 ng/spot; correlation coefficient (r) was 0.9995. The low values of limit of detection and limit of quantification (12.4 and 37.5 ng/spot, respectively) have demonstrated the sensitivity of the developed method. The reported method was precise in both intra-day as well as inter-day analysis; % RSD of peak area was found to be less than 2%, and has an accuracy within 100 ± 2%. The developed method has a potential to quantify zanamivir from its diskhaler formulation without any interference from other components. The applicability of the method was demonstrated by excellent recovery of analyte (99.8%) from diskhaler formulation. The current analytical method can be applied for routine analysis of zanamivir in pure form and pharmaceutical formulation in quality control laboratories.