This work presents a valuable contribution to analytical chemistry field as well as those related disciplines including pharmacology, biochemistry and medicine, principally. A new application of the CPE for the analysis of ARV in human plasma by UFLC-MS/MS was proposed for direct application in real studies.The CPE-UFLC-MS/MS was developed on real matrices, which allowed a comprehensive characterization and optimization of the procedure with a feasible validation according to international standards. The proposed analytical methodology was developed and applied for analyzing of ABC, EFV, 3 TC and NFV in human plasma. Under optimized working conditions the proposed analytical methodology meets standards requirements of international guidelines, which makes it suitable for pharmacokinetic studies of four ARV, as well as for therapeutic monitoring of HIV patients. Additionally to its analytical performance, it is important to point out that the proposed sample preparation technique has an environmentally friendly character that makes it suitable for current green chemistry trends.