It recently became benzodiazepine abuse a serious social problem in the world, and the Chromatographic applications in the pharmaceutical sector and drugs among the fastest in fields and science detection of toxins in the blood. Using a simple and convenient method by High-Performance Liquid Chromatography (HPLC) high-tech (Agilent technologies1200series) to detect abuse of benzodiazepines in biologic samples is necessary to predict and protect human health risks. It has developed many of the helpful methods for qualification and quantification of medically prescribed benzodiazepines, in addition to many analytical techniques to determine the quantities of isolation and benzodiazepines in biological samples has already been published; among them, each thin-layer chromatography (TLC) and immunoassay approach remain extremely useful for a first rapid screening. However, both techniques lack high specificity and in the case of immuno-analysis, it is no discrimination between parent’s benzodiazepines and metabolites, On the other hand, gas chromatography’s (GC) with flame-ionization (GC/FID) or mass spectrometric detection (GC/MS) are much more sensitive and specific, but they require complex equipment and privatization procedure. Otherwise, the GC process can decompose thermolabile benzodiazepines like oxazepam and chlorazepate; the above-mentioned reasons resulted in a still-increasing popularity of HPLC for screening and quantitation of benzodiazepines in bio samples. Especially it is considered one of the most widely used analytical tools in the biotech industry. In this current study, we review the psychological description of this methodology (Agilent1200HPLC). The analytical results have been grouped based on the type of analysis narcotic drugs of the benzodiazepine group, of Schedule IV according to the rankings. These drug is; Alprazolam, Diazepam, Estazolam. To aim to know the concentration values of these drugs in serum in the hours varying after a dose medication when users abuse, it was been used three rabbits for the experiments work. The drug were given to rabbits, each one separately and drug dose as follows;(alprazolam, diazepam and estazolam), was extracted biological samples (serum) from the blood of rabbits in different hours;(alprazolam:1h,2h,4h,8h,12h,24h),(diazepam:1h,2h,10h,24h,36h),(estazolam:1h,3h,8h,12h,24h) respectively.Basic calibration standard to prepare of reagent is (KH2PO4[8g]-K2HPO4.3H2O [2g]-H2O [1000ml]), and has melted all of drugs in a solution consisting of (HCL) and (H2O), added by1:1. Moreover, separated was conducted on a carbon column (15Cm)(4.6×150mm,5μm)(C18) stationary phase, and are used methanol (CH3OH) and distilled water (H2O), and the mobile phases are (PBS:CH3OH) at a flow rate was (1ml/min), and an angel is (λ=238nm). The chemical residues from the device during the experiments are (CH3CN),(H2O).It has been injected of samples are as follows; Alprazolam (10μl), Diazepam (5μl), Estazolam (6μl,8μl,5μl) in (1h,3h,8h,12h,24h) respectively. Moreover, injection volumes of three serum samples are (10,7, and5μl), directly in the column with a mobile phase (PBS:CH3OH), and used UV to detect for alprazolam (λ=254nm), diazepam (λ=254nm), and estazolam (λ=223nm).In the results, the minimum concentration of alprazolam in a serum sample equivalent was (0μg/L) at (24h), while the maximum concentrations was (404.45μg/L) at (2h) in detection. In addition, the minimum concentration of diazepam was (141μg/L) at (36h), while the maximum concentration was (584μg/L) at (2h). In addition, estazolam, the minimum concentration was (27.2μg/L) at (24h), while the maximum concentration was (71.8μg/L) at (3h). Each detection process is over in20minutes, and then showed the tables and curves.