1-(3,4-Dimethoxyphenyl)-4-methyl-5-ethyl-7,8-dimethoxy-5H-2,3-benzodiazepine (tofisopam) contains one chiral center, so two enantiomeric forms exist. The ring system of tofisopam possesses two sterically stable boat structures, leading to two distinct conformers for each enantiomer. A method was developed for the separation of these enantiomers and conformers in the drug substances and drug products. Separation was achieved with a separation factor of at least 3.9 for any adjacent peaks. Validation of the method challenged linearity, limit of detection, limit of quantification, specificity, accuracy, repeatability, intermediate precision, robustness, and stability of standard and sample solutions, and all validation results met the acceptance criteria. A study of accuracy at 80%, 100%, and 120% levels gave recoveries of 100 +/- 1%. The RSD of six sample injections for repeatability was less than 0.5%. The detection limit of tofisopam enantiomer was as low as 0.12 microg/mL. The kinetics and thermodynamics of the interconversion of tofisopam conformers were also investigated, and the kinetic and equilibrium constants of the interconversion process were determined at 15 degrees C, 25 degrees C, and 35 degrees C.