Context: In the development of a good analytical method, the selection of appropriate conditions for quantifying drugs is essential. The method validation should be determined before conducting the dissolution test. Aims: To develop and validate two analytical methods by UV spectrophotometry to quantify lamivudine and zidovudine in dissolution test. Methods: The dissolution conditions were 900 mL of dissolution medium (pH 1.2, pH 4.5, and pH 6.8), using paddles, at 75 rpm, and sampling time points of 5, 10, 15, 20, and 30 minutes. The analytical methods were developed by scanning analytical solutions in the UV region. The proposed methods were validated in accordance with the ICH guideline and the United States Pharmacopoeia. Results: Lamivudine showed wavelengths at 280 nm, 273 nm, and 270 nm in buffers at pH 1.2, pH 4.5, and pH 6.8, respectively; while zidovudine showed a wavelength at 266 nm in all three buffers. Regression analysis confirmed linearity (r2>0.998). Placebos and diluents showed no analytical interference. LOD and LOQ were lower than the linearity range. Recovery percentages were within 95% to 105%. RSD values were below 2% and 7% for repeatability and intermediate precision, respectively. No changes introduced modified the method response. The analytical solutions were stable until 24 hours. The results showed that Whatman No 41, No 42, and PVDF 0.45 µm filters can be used. Conclusions: The proposed analytical methods demonstrated linearity, specificity, accuracy, and precision. In addition, LOD and LOQ, robustness, stability of the analytical solution, and filter test showed satisfactory results.