The human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) pandemic continues to adversely affect human health and life. Although several classes of HIV treatment drugs (antiretrovirals, ARVs) are available, there is a continuing need for new drug modalities, due to inferior drug safety profiles and development of virus resistance. A potential novel class of ARVs are the maturation inhibitors (MIs), which inhibit the ultimate step of HIV-1 protease cleavage during final virus assembly, resulting in immature virus particles incapable of productively infecting other cells. To support ongoing studies on MIs that focus on the biochemical process at the mechanistic level, a liquid chromatography-mass spectrometric (LC-MS) based in vitro assay was developed to quantitatively characterize the HIV-1 protease driven rate of cleavage of HIV-1 virus-like particles (VLPs, fully assembled immature HIV-1 Gag polyprotein), thereby affording kinetic cleavage profiles. Utilizing surrogate analyte methodology on proteinaceous analytes and high resolution accurate mass analysis allowed for the multidimensional design of experiments affording quality data on variables such as Gag (VLP) polymorphic variants, structure–activity-relationship (SAR) of MIs, reagent concentrations, and sampling times. The unique approach of data normalization to an assay internal proteolytic analyte as a control standard eliminated the need for external stable isotope labeled analytes but nevertheless resulted in precise bioanalytical assay data utilized in comparative MIs inhibitory studies.