Capsidiol is a bicyclic sesquiterpene, which accumulates extracellularly in plants, and has been isolated from many types of Solanaceae. It acts as a phytoalexin produced by Nicotiana tabacum in response to pathogens. Capsidiol has antifungal activity and is formed first in tobacco and pepper plants after infestation. The amount of capsidiol in tobacco cell suspension culture has been previously determined by solid-phase extraction and organic solvent extraction with thin-layer chromatography or gas chromatography analysis. A high-performance liquid chromatography method with UV detection at 210 nm on a C(8) column utilizing both extraction methods was developed to analyze capsidiol in suspension cell culture. The HPLC method was linear in the concentration range of 0.1-2.0 mg/L. The lower limit of quantitation was 0.1 mg/L. Organic solvent extraction and solid-phase extraction methods were compared. Both methods are generally similar in their overall efficiency (82% and 75%, respectively), but eliminations of interfering compounds are different. The relative standard deviation across five extractions of known amounts of capsidiol from plant sample was less than 5.1%. The relative standard deviation across five elicitations of cell cultures was less than 5.9%. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analysis of capsidiol was performed, and corresponding mass spectra are presented.