AbstractExtracellular vesicles (EVs), crucial mediators of cell‐to‐cell communication, hold significant diagnostic potential due to their ability to concentrate protein biomarkers in bodily fluids. However, challenges in isolating EVs from biological specimens hinder their widespread use. The preferred strategy involves direct analysis, integrating isolation and analysis solutions, with immunoaffinity methods currently dominating. Yet, the heterogeneous nature of EVs poses challenges, as proposed markers may not be as universally present as thought, raising concerns about biomarker screening reliability. This issue extends to EV‐mimics, where conventional methods may lack applicability. Addressing these challenges, the study reports on Membrane Sensing Peptides (MSP) as pan‐vesicular affinity ligands for both EVs and their non‐canonical analogs, streamlining capture and phenotyping through Single Molecule Array (SiMoA). MSP ligands enable direct analysis of circulating EVs, eliminating the need for prior isolation. Demonstrating clinical translation, MSP technology detects an EV‐associated epitope signature in serum and plasma, distinguishing myocardial infarction from stable angina. Additionally, MSP allow analysis of tetraspanin‐lacking Red Blood Cell‐derived EVs, overcoming limitations associated with antibody‐based methods. Overall, the work underlines the value of MSP as complementary tools to antibodies, advancing EV analysis for clinical diagnostics and beyond, and marking the first‐ever peptide‐based application in SiMoA technology.