Besides its many pharmacodynamic actions, the pyridoindole stobadine was found to exert antioxidant activity and thus possesses the potential to protect various tissues against oxidative stress. This overview is focussed on both the evaluation of the chemical procedures used in the bioassay of stobadine and its metabolites and on the comparison of their quality in the light of applicability for preclinical and clinical pharmacokinetic experiments. All methods and applications were performed at the Institute of Experimental Pharmacology, SASc in Bratislava, Slovakia. In pharmacokinetic and toxicokinetic studies, [3H]-labeled stobadine dihydrochloride was administered intravenously or orally to rats in single and repeated doses. Liquid-liquid extraction was used for selective isolation of stobadine and its metabolites from biological matrix, followed by liquid scintillation quantification. A TLRC method was developed both to check the radiochemical purity of [3H]-stobadine and to quantify the labeled drug in rat plasma. A spectrofluorometric approach was used for determination of stobadine in dog serum and urine after its administration in the form of either the dihydrochloride or the dipalmitate. The method allowed us to perform a bioavailability study and a long-term toxicological study. The HPLC method with a limit of detection of 10 ng/ml of plasma proved suitable for calculating the compartmental pharmacokinetic parameters of both salt forms of stobadine administered to dog and man. This method was based on solid-phase extraction procedure by using Separcol SI C18 cartridges. In a GC method, the combination of capillary column separation and nitrogen-specific detection permitted the assay of serum stobadine concentrations as low as 5 ng/ml. The detection limit of the GC/MS method was 1 ng/ml of plasma or of phosphate buffer saline. This method was used for a bioequivalence study of two stobadine dipalmitate dosage forms and for a transdermal penetration study of stobadine acyl derivatives. All the developed assays proved to be appropriate for low-concentration determination of stobadine in a wide range of pharmacokinetic studies.