Understanding the pharmacology of drugs in breast milk is important for the health of both mother and baby. Current methods to measure drug concentrations in breast milk are not easily validated for precision or accuracy, primarily because of suboptimal sample clean-up. We report here an optimized clean-up method to remove both proteins and fat from milk, thereby enhancing the extraction efficiency of antiretroviral drugs. Recoveries were consistently above 91% for all drugs, demonstrating that this method successfully and reliably released drugs from fat globules. With use of 200 μL of human breast milk, an high-performance liquid chromatography/ultraviolet method for simultaneously detecting lamivudine, stavudine, zidovudine, and nevirapine was validated over the range of 20 to 20,000 ng/mL. Intra- and interday precision (average percent relative standard deviation) and accuracy (average percent deviation from nominal) was less than 3.6% and 7.5%, respectively. Intra- and interday accuracy (average percent deviation from nominal) was less than 0.25% and 1.3%, respectively. This novel method efficiently, reliably, and accurately measured antiretroviral drugs in breast milk and can be applied to any matrix containing fat and protein.