Bacterial membrane vesicles (BMVs) are small, spherical structures released by Gram-positive and Gram-negative bacteria that play essential roles in intercellular communication, nutrient acquisition, and antibiotic resistance. BMVs typically range from 40 to 400 nm in diameter and contain a single membrane derived from the bacterial membrane, comprising proteins, lipids, nucleic acids, and other biomolecules. Notably, the molecules located on the surface of BMVs facilitate interactions with neighboring cells, including the transfer of functional genes, coordination of bacterial growth through quorum sensing, and delivery of toxins during infections. In addition, BMVs exhibit heterogeneity in their surface composition, which influences their interactions with host and bacterial cells. It is therefore essential to understand not just the composition of BMVs, but the localization of the molecules of interest, particularly those on the surface. In this chapter, we describe several methods that can be used to quantify and characterize the protein and nucleic acid composition, particularly on the surface of BMVs. We describe quantitative immunoblot and ELISA protocols that enable quantification of the concentration of a particular protein of interest. We also describe an enzymatic digestion protocol to determine whether the protein of interest is located on the surface or within the lumen of the BMV, as well as a nucleic acid staining procedure that enables quantification of dsDNA specifically located on the surface of the BMVs. Together, these tools provide a detailed analysis of the protein and nucleic acid composition of BMVs that can be further combined with various separation techniques to study variations within different populations.