A HPTLC method for the quantitative analysis of nicotine present in the various commercial tobacco preparations has been developed. Methanol extract of the formulations were analyzed on a silica gel 60 GF254 TLC plates with spot visualization under UV and scanning at 235 nm in absorption-reflection mode. Calibration curves were found to be linear in the range 2μg-25 μg. The Correlation coefficients were found to be 0.991 for nicotine. The average recovery rate was 95% (CV% 1.35) for nicotine. From the present study LOD was determined as 0.08 μg/spot for Nicotine. The validity of the method with respect to analysis was confirmed by comparing the UV spectra of the commercial tobacco preparation with that of the standard within the same Rf window. © 2012 by Nova Science Publishers, Inc. All rights reserved.