Combined therapy for HIV treatment shows superior efficacy in comparison to single therapy. Furthermore, the separation and determination of the combined dosage forms hold a significant place in the pharmaceutical industry. Not only reverse phase high-performance liquid chromatography (HPLC) but also hydrophilic interaction liquid chromatography (HILIC) can be used for these purposes. Contrary to conventional RP systems, HILIC may be an alternative for the analysis of polar substances. The aim of this study was the step-by-step development of a simple, rapid, and reliable method for the simultaneous determination of antiviral compounds from their marketed formulation (Trizivir). In order to achieve this goal, various mobile phase systems, buffer types, and concentrations were prepared to provide an appropriate separation. Different types of columns were tested to find the best resolution and high efficiency for the studied compounds. The proposed method provided a simple procedure for the simultaneous analysis of abacavir, lamivudine, and zidovudine in their pharmaceutical preparation within approximately 2 min using the conventional HPLC system. The developed method was fully validated according to the International Council for Harmonisation guidelines from the viewpoint of selectivity, sensitivity, precision, accuracy, linearity, limit of detection, and quantification.