This study investigates the distinct RNA profiles in subpopulations of extracellular vesicles (EVs) derived from apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). EVs were isolated from three different cell lines—HMC-1, TF-1, and BV-2—using two centrifugation-based protocols. The RNA profiles were analyzed using a Bioanalyzer, and the morphology of the vesicles was visualized by transmission electron microscopy (TEM). The results show that ribosomal RNA (rRNA) is primarily detectable in ABs, while smaller RNAs without prominent rRNA peaks are found in EXOs. MVs contained little or no RNA, except for those from TF-1 cells. TEM analysis confirmed the presence of ABs, MVs, and EXOs in the respective vesicle fractions. Flow cytometry revealed the presence of CD63 and CD81 on all vesicle fractions, except for CD9 in TF-1-derived vesicles. The study demonstrates that centrifugation-based protocols are effective for distinguishing subpopulations of EVs and highlights the importance of considering the RNA profiles when studying the functionality of RNA in different vesicles.This study investigates the distinct RNA profiles in subpopulations of extracellular vesicles (EVs) derived from apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). EVs were isolated from three different cell lines—HMC-1, TF-1, and BV-2—using two centrifugation-based protocols. The RNA profiles were analyzed using a Bioanalyzer, and the morphology of the vesicles was visualized by transmission electron microscopy (TEM). The results show that ribosomal RNA (rRNA) is primarily detectable in ABs, while smaller RNAs without prominent rRNA peaks are found in EXOs. MVs contained little or no RNA, except for those from TF-1 cells. TEM analysis confirmed the presence of ABs, MVs, and EXOs in the respective vesicle fractions. Flow cytometry revealed the presence of CD63 and CD81 on all vesicle fractions, except for CD9 in TF-1-derived vesicles. The study demonstrates that centrifugation-based protocols are effective for distinguishing subpopulations of EVs and highlights the importance of considering the RNA profiles when studying the functionality of RNA in different vesicles.