A HPTLC method has been developed and validated for the determination of tea tree oil from cosmeceutical formulations. Tea tree oil concentration was estimated by analyzing the terpinen-4-ol content. The method employed TLC aluminium plates precoated with silica gel 60F-254. The solvent system consisted of toluene and ethyl acetate in the ratio 85:15. The calibration curve of terpinen-4-ol was linear in the range of 100-900 ng. The polynomial regression data for the calibration plots showed a good linear relationship with r(2)=0.9949. The Rf value of terpinen-4-ol was found to be 0.62+/-0.05. The method was validated for precision and accuracy. The minimum detectable amount was found to be 60 ng. The limit of quantitation was found to be 100 ng. The drug content was within the limits (+/-5% of the labeled content of the formulations). The recovery of tea tree oil was greater than 99%. The method was found to be simple, sensitive, precise, accurate and specific for estimation of tea tree oil from formulations.