Atherosclerosis is a complex multifactorial disease characterised by the build-up of plaque in the arterial wall. Apolipoprotein E gene-deficient (Apoe–/–) mice serve as a commonly used tool to elucidate the pathophysiology of atherosclerosis due to their propensity to spontaneously develop arterial lesions. To date, however, an integrated omics assessment of atherosclerotic lesions in individual Apoe–/– mice has been challenging due to the small amount of diseased and non-diseased tissue available. To address this current limitation we developed a multi-omics method (Multi-ABLE) based on the proteomic method called Accelerated Barocycler Lysis and Extraction (ABLE) to assess the depth of information that can be obtained from arterial tissue derived from a single mouse by splitting ABLE to allow for a combined proteomics-metabolomics-lipidomics analysis (Multi-ABLE). The new method includes tissue lysis using pressure cycling technology (PCT) in a Barocycler, followed by proteomic analysis of half the sample by nanoLC-MS, and sequential extraction of lipids (organic extract) and metabolites (aqueous extract) combined with HILIC and reversed phase chromatography and time-of-flight mass spectrometry on the other half. Proteomic analysis identified 845 proteins, 93 of which were significantly altered in lesion-containing arteries. Lipidomic and metabolomic analyses detected around 5,000 lipid and 400 metabolite features, including several that were significantly altered in diseased tissue. The Multi-ABLE method is the first to apply a concurrent multi-omics pipeline to cardiovascular disease using small (<5 mg) tissue samples, and will be applicable to other diseases where limited size samples are available at specific points during disease progression.