Atherosclerotic cardiovascular disease is the leading cause of death worldwide. For decades, mouse modeling of atherosclerosis has been the mainstay for preclinical testing of genetic and pharmacological intervention. Mouse models of atherosclerosis depend on supraphysiological levels of circulating cholesterol carried in lipoprotein particles. Lipoprotein particles vary in atherogenicity, and it is critical to monitor lipoprotein levels during preclinical interventions in mice. Unfortunately, the small plasma volumes typically harvested during preclinical experiments limit analyses to measuring total cholesterol and triglyceride levels. Here we developed a high-throughput, low-cost targeted multiple reaction monitoring (MRM) stable isotope dilution (SID) mass spectrometry assay for simultaneous relative quantification of nine apolipoproteins using a few microliters of mouse plasma. We applied the MRM assay to investigate the plasma apolipoproteome of two atherosclerosis models: the widely used ApoE knockout model and the emerging recombinant adeno-associated virus-mediated hepatic Pcsk9 overexpression model. By applying the assay on size-exclusion chromatography-separated plasma pools, we provide in-depth characterization of apolipoprotein distribution across lipoprotein species in these models, and finally, we use the assay to quantify apolipoprotein deposition in mouse atherosclerotic plaques. Taken together, we report development and application of an MRM assay that can be adopted by fellow researchers to monitor the mouse plasma apolipoproteome during preclinical investigations.