Human immunodeficiency virus (HIV) is a global health concern, and the development of effective methods for the detection and quantification of antiretroviral drugs is crucial for monitoring therapeutic efficacy and patient compliance. Herein, we present a novel approach utilizing graphene quantum dots (GQDs) for the determination of abacavir, a widely used nucleoside reverse transcriptase inhibitor in the treatment of HIV. The sensing mechanism was investigated through Stern-Volmer analysis, thermodynamics studies, and density functional theory calculations, which revealed the strong binding interactions between GQDs and abacavir via a static quenching process. Factors affecting the analytical performance, such as pH, GQDs concentration, and incubation time, were systematically optimized to achieve a linear detection range of 100 to 1000 ng/mL with a low detection limit of 17.49 ng/mL. The method was validated in accordance with ICH guidelines demonstrating excellent linearity, accuracy, precision, robustness and selectivity making it suitable for the quantification of abacavir in pharmaceutical formulations and biological samples. The method also demonstrated higher sensitivity and more environmentally friendly characteristics when compared to previously reported chromatographic techniques, showcasing the potential of GQDs as a superior alternative for the traditional detection approaches of pharmaceutical compounds.