Lopinavir, a widely used antiretroviral protease inhibitor, suffers from poor aqueous solubility and low oral bioavailability, limiting its therapeutic potential. This study aimed to develop and optimize Lopinavir-loaded nanostructured lipid carriers (NLCs) using a Design of Experiments (DoE)-based approach. Lipid matrices were prepared using a blend of solid (glyceryl monostearate) and liquid (oleic acid) lipids stabilized with polysorbate 80 as a surfactant. A central composite design (CCD) was employed to evaluate the influence of lipid ratio, surfactant concentration, and sonication time on key responses—particle size, polydispersity index (PDI), zeta potential, entrapment efficiency (EE%), and drug release. Optimized NLCs exhibited a mean particle size of 142.3 ± 4.2 nm, PDI of 0.21 ± 0.03, zeta potential of −28.4 ± 1.8 mV, and EE% of 89.2 ± 2.6%. In vitro release studies showed a sustained release profile over 24 hours. The study demonstrates that DoE-guided optimization is an efficient strategy for developing stable Lopinavir-loaded NLCs with enhanced physicochemical properties, offering improved drug delivery for antiretroviral therapy.