Theobromine is the primary methylxanthine found in chocolate, a common cigarette additive. A simple and reproducible method has been developed and validated for the analysis of theobromine in processed tobacco by using high performance liquid chromatography (HPLC). Theobromine is extracted from tobacco sample with water by sonication. The aqueous extract is analyzed by HPLC with a propyl-linked pentafluoropheny (PFP) column using methanol/water/acetic acid (10/89/1, v/v/v) as the mobile phase at 0.8 mL/min and a ultra-violet (UV) detector at 280 nm. The utilization of a PFP column allows for better separation of tobacco matrix interference from the theobromine peak, compared to the most commonly used C18 reverse phase column. The recovery of theobromine from spiked tobacco sample is 97% with relative standard deviation under 6%. The detection limit of theobromine is 0.38 μg/g in tobacco. This method was applied to several commercial tobacco products, of which the concentration range of theobromine varied from below the detection limit to 86.5 μg/g.