Antiretroviral therapy, including Lamivudine (LMV), Dolutegravir (DTR), and Tenofovir disoproxil fumarate (TDF), plays a crucial role in managing HIV/AIDS and chronic hepatitis B virus. The reported methods for analyzing these drugs face challenges related to environmental sustainability. Existing analytical techniques, such as HPLC and LC-MS, rely heavily on organic solvents, posing ecological and health concerns. A novel eco-friendly micellar liquid chromatography (MLC) approach was developed to determine LMV, DTR, and TDF in pharmaceutical formulations concurrently. Different variables were considered, such as the micellar mobile phase’s composition, sodium lauryl sulfate (SLS), or Polyoxyethylene(23)lauryl ether (Brij-35), in addition to the pH value. The optimized mobile solution consisted of SLS (0.1 M) and Brij-35 (0.02 M) in water, which was adjusted to pH 3.5 and at an elution rate of 1 mL min−1 using Symmetry C18 (100 × 4.6 mm, 3.5 µm) at a temperature of 40 °C. The examined drugs were separated in less than 8 min and performed through a UV detector at 258 nm. The developed approach showed linearity across concentrations of 7.5–150, 5–100, and 15–300 µg mL−1 for LMV, DTR, and TDF, respectively. The percentage recoveries ranged from 97.5 % to 100.8 % for drugs. Different green assessment tools, like AGREE metrics, analytical eco-scale, and green analytical procedure index, were used to investigate the greenness score. The proposed method achieved superior sustainability compared to reported methods, scoring 92 on the analytical eco scale. The proposed MLC method is a sustainable, accurate, and efficient analytical technique to assess LMV, DTR, and TDF in pharmaceutical formulations. It complies with the GAC principles and is suitable for routine quality control with minimal environmental impact.