Extracellular vesicles (EVs) laden with lipids, proteins, DNA, and micro-RNAs play important biological functions in intercellular communication and have pivotal roles in pathophysiological conditions. Characterization of the EVs has always been a multistep process involving large volumes, and they are heterogeneous in size and properties. A multitude of approaches is used to distinguish the EVs. Here, we report simple citrate reduced silver nanoparticles assisted surface-enhanced Raman spectroscopy (SERS) as a tool to distinguish EVs extracted from several cell lines isolated under autophagic conditions (nitrogen starvation). This study is the first report of its kind in characterizing EVs from cells under autophagic conditions using SERS. We used two cancerous cell lines, HeLa, its corresponding autophagy-deficient cell line (Atg5-/-), and a noncancerous cell line, HEK293, to isolate EVs. Our study helps in the facile detection and differentiation of EVs isolated between two closely related human cell lines that differ by their autophagic ability. The principal component analysis (PCA) of the SERS spectra of these EVs consistently showed the presence of distinct chemical compositions of the EVs. SERS of EVs can help in probing more into the molecular level information from EVs and could become a powerful tool once coupled with improved microscopy techniques for diagnosis and therapy.