Alzheimer's is a neurodegenerative disease. Its symptoms are attributed to a deficiency of cholinergic neurotransmission. The drugs of choice for the treatment of Alzheimer's disease are acetylcholinesterase (AChE) inhibitors. Starting in the 1980's from non-specific AChE inhibitors, the first-generation drugs such as physostigmine, a second generation of more selective and better tolerated products has been developed. Methods to detect and quantify these drugs and their metabolites in biological samples have been developed for analysis in plasma, blood, urine and cerebrospinal fluid. Diverse detection techniques have been used, such as ultraviolet, fluorescence, electrochemical and mass spectrometry. In this review, the methods applied to the analysis of these drugs and their metabolites in different biological matrices are reviewed and discussed. The stability of these drugs in biological matrices and under stress-conditions is also included in the discussion. © 2010 John Wiley & Sons, Ltd.