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Anxiety disorders are among the most prevalent mental disorders in the general population. Nearly 30 million persons are affected in the United States, with women affected nearly twice as frequently as men. These disorders are frequently treated using benzodiazepines (Diazepam) and non-benzodiazepines (Zaleplon). Non-benzodiazepines have replaced the benzodiazepines for anxiety treatment due to low risk of dependence and CNS effects encouraging drug abuse. This work was designed to investigate the toxicological overdose effect of zaleplon and diazepam on behavior, blood pressure, heart rate, brain neurotransmitter content and liver & kidney functions in rats. Rats were divided into five groups: 1% Tween 80, p.o. (control), diazepam (1 & 4 mg/kg, p.o.) and zaleplon (1 & 4 mg/kg, p.o.). All groups were injected daily for four weeks to perform the following: behavioral study (assessment of motor co-ordination and anxiety performance), measurement of blood pressure & heart rate, determination of the free amino acid and monoamine contents in the whole brain, determination of serum aminotransferases activity, total protein, total and direct billirubin, creatinine & blood urea nitrogen of experimental rats. Zaleplon decreased motor co-ordination, blood pressure and heart rate with extent lesser than diazepam. However, it decreased anxiety performance and brain monoamine content with extent more than diazepam. While, diazepam increased brain amino acid content, serum aminotransferases activity, total protein, total & direct billirubin, creatinine and blood urea nitrogen by magnitude more than zaleplon.