Abstract: A method of HPLC for the determination of nicotine in various parts of tobacco with hydrophilic chromatographic separation was proposed. The sample was separated on Atlantis HILIC chromatographic column (150 mm×4.6 mm, 5 μm), and a mixture of acetonitile, 10 mmol·L-1 ammonium acetate solution and glacial acetic acid (72+28+2) was used as mobile phase. UV-detection at 260 nm was adopted in the determination. Linear relationship between values of peak area and mass concentration of nicotine was kept in the range of 0.001-0.1 g·L-1, with detection limit (3S/N) of 0.17 mg·L-1. Tests for recovery were made by addition of standard solutions at 3 concentration levels, giving values of recovery in the range of 98.2%-98.9% with RSD′s (n=6) less than 2.0%. Samples were taken from upper, middle and lower part of the tobacco leaves as well as from the stems, and analyzed by the proposed method, the contents of nicotine were found to decrease in the respective order.