Exosomes are extracellular vesicles derived from the endosomal compartment. They contain a multitude of bioactive constituents and mediate intercellular communication in health and disease. The frequently used biomarkers of exosomes are heterogeneous and do not exhibit universal utility across different cell types. To uncover ubiquitous and abundant proteins that can serve as universal biomarkers of exosomes, we employed an unbiased and quantitative proteomic approach based on Super SILAC coupled to high-resolution mass spectrometry (MS). In total 1,243 proteins were consistently quantified in the proteome of exosomes, irrespective of the cellular source or isolation method. Among them, a cohort of 22 proteins were universally enriched, and 15 proteins consistently depleted in the proteome of exosomes when compared to cells. Among the enriched proteins, we found several membrane proteins and GTPases, whereas the cohort of depleted proteins was predominantly composed by nuclear proteins. Our study revealed that Syntenin-1 is the most abundant ubiquitous protein in exosomes from distinct cell sources, thus representing an ideal candidate for universal biomarker of exosomes.