A high-performance liquid chromatographic technique was developed to quantitate the major alkaloids from both fresh green tissue and air dried tobacco leaf tissue. The procedure involves an aqueous extraction of the milled tissue followed by separation of the alkaloids on a reversed-phase Cl8 column with a mobile phase of 40 % methanol containing 0.2 % phosphoric acid buffered to pH 7.25 with triethylamine. This procedure provides quantitative analysis of the four major alkaloids in tobacco and can be partially automated to handle large numbers of samples.