Physostigmine is a major alkaloid found in the seeds of the fabaceous plant Physostigma venenosum. It is a powerful and reversible acetylcholine esterase inhibitor which effectively increases the concentration of acetylcholine at the sites of cholinergic transmission. It exerts its cholinesterase inhibitor effect in both the periphery and central nervous system. Many studies on physostigmine have involved the reliance on techniques that extract and quantify physostigmine in biological samples. This paper presents an overview of the currently applied methodologies for the determination of physostigmine and its metabolites in various biological samples. Papers published from January 1980 to December 2003 were taken into consideration for the discussion of the metabolism and analytical method of physostigmine. HPLC methods have been discussed and used in most of the references cited in this review. A few CE and RIA methods that have been recently reported are also mentioned in this paper. Basic information about the sample assayed, sample preparation, chromatographic column, mobile phase, detection mode and validation data are summarized in a table.