This systematic review analyzes 471 extracellular vesicle (EV)-related clinical trials, highlighting trends in clinical applications and methodologies, with a focus on EV subpopulations. Diagnostics and companion diagnostics represent the majority of trials, with cancer being the most common application. Therapeutic trials primarily use mesenchymal stromal cell (MSC) EVs for respiratory diseases. Common isolation and characterization techniques include ultracentrifugation and RNA-sequencing, though detailed methodology is often not reported. Most trials use bulk EV characterization, with only 11% incorporating EV subpopulations. This highlights the need for better methods to study EV subpopulations, which could improve translational efforts. The review emphasizes the importance of detailed methodological reporting and the potential of EV subpopulations to enhance the success of EV-based interventions. It also notes the growing interest in EVs for clinical applications, with a need for standardized methodologies, improved characterization techniques, and validation studies to ensure the efficacy and safety of EV-based therapies. The study underscores the importance of addressing methodological gaps and focusing on EV subpopulations to advance personalized medicine.This systematic review analyzes 471 extracellular vesicle (EV)-related clinical trials, highlighting trends in clinical applications and methodologies, with a focus on EV subpopulations. Diagnostics and companion diagnostics represent the majority of trials, with cancer being the most common application. Therapeutic trials primarily use mesenchymal stromal cell (MSC) EVs for respiratory diseases. Common isolation and characterization techniques include ultracentrifugation and RNA-sequencing, though detailed methodology is often not reported. Most trials use bulk EV characterization, with only 11% incorporating EV subpopulations. This highlights the need for better methods to study EV subpopulations, which could improve translational efforts. The review emphasizes the importance of detailed methodological reporting and the potential of EV subpopulations to enhance the success of EV-based interventions. It also notes the growing interest in EVs for clinical applications, with a need for standardized methodologies, improved characterization techniques, and validation studies to ensure the efficacy and safety of EV-based therapies. The study underscores the importance of addressing methodological gaps and focusing on EV subpopulations to advance personalized medicine.