THE PRESENT study described a novel application of simple potentiometric biosensors for analysis of anti-cholinesterase (Alzheimer) drugs in vitro. The proposed method was based on inhibition of acetylcholineesterase enzyme (AChE) by Alzheimer drugs namely; donepezil (DOP) and galantamine (GAL) in addition to biperdien (BP) and ipratropium (IPBr). Based on the relative inhibition action of drug on AChE activity, different sensitivities were recorded ranging between 0 to 18.0 ng, 0 to 90.0 ng, 0 to 4.69 μg and 0 to 9.96 μg mL-1for the aforementioned drugs in the same order depending on the nature of drugs and their corresponding LD50 values. The proposed procedures were successfully applied for determination of drugs in pharmaceutical formulation and biological samples with sensitivity and accuracy comparable with the official method. The presented approach can be suggested for testing of pharmaceutical preparation toxicity against cholinesterase enzymes in vitro.