High performance liquid chromatography (HPLC) has become one of the most important and fastest growing techniques in analytical laboratory. The basic theory behind high performance liquid chromatography is not new, but its development is fast now by introduction of new packing materials and columns, which allow the chromatographer to perform separations faster and with greater resolution than had previously been attainable. In this thesis HPLC separations will be discussed and applied to some important drugs and drugs mixtures. This thesis consists of the following chapters: Chapter І: Introduction The introduction is subdivided into two sections: 1) General introduction to HPLC-components, mechanisms of separation and discussing some of the parameters affecting separation. 2) Introduction about the drugs chosen for investigation. Chapter ІІ: Study of effect of different chromatographic variables on separation of two drugs mixtures that are already formulated in the market or expected to be formulated in the near future: 1) Comparison between conventional C18 stationary phase and calixarene stationary phase on separation of group of antihypertensive drugs including Hydrochlorothiazide, Losartan potassium, Valsartan, Irbesartan, Telmisartan, Spironolactone and Xipamide. 2) Study of chromatographic behavior of Sildenafil citrate, Tadalafil, Apomorphine hydrochloride and Tramadol hydrochloride on C18 stationary phase. Chapter ІІІ: Quantitative HPLC-determination of two mixtures of drugs using validated methods that provide enhanced separation and faster analysis time: 1) Chromatographic determination of Hydrochlorothiazide, Losartan potassium, Irbesartan and Valsartan using C18 column. 2) Chromatographic determination of Apomorphine hydrochloride, Sildenafil citrate and Tadalafil using calixarene stationary phase. Chapter ІV: Quantitative and validated new stability indicating HPLC methods for determination of: 1) Praziquantel in bulk powder and pharmaceutical preparation. 2) Atenolol and Nifedipine mixture in bulk powders and pharmaceutical preparation.