ConspectusMicrofluidic technologies have become a highly effective platform for the precise and reproducible production of nanovesicles used in drug and nucleic acid delivery. One of their key advantages lies in the one-step assembly of multidrug delivery nanovesicles, which improves batch-to-batch reproducibility by minimizing the intermediate steps typically required in conventional methods. These steps often involve complex hydrophobic and electrostatic interactions, leading to variability in the nanovesicle composition and performance. Microfluidic systems streamline the encapsulation of diverse therapeutic agents, including hydrophilic nucleic acids, proteins, and both hydrophobic and hydrophilic small molecules, within a single chip, ensuring a more consistent production process. This capability enables the codelivery of multiple drugs targeting different disease pathways, which is particularly valuable in reducing the risk of drug resistance.Despite the promise of nanovesicles for nucleic acid delivery, their clinical translation has been hindered by safety concerns, particularly cytotoxicity, which has overshadowed efforts to improve in vivo stability and delivery efficiency. Positively charged nanovesicles, commonly used to encapsulate negatively charged nucleic acids, tend to exhibit significant cytotoxicity. To address this, charge-shifting materials that respond to pH changes or surface modifications have been proposed as promising strategies. Shifting the surface charge from positive to neutral or negative at physiological pH can reduce the cytotoxicity, enhancing the clinical feasibility of these nanovesicle-based therapies.Microfluidic platforms offer precise control over key nanovesicle properties, including particle size, rigidity, morphology, and encapsulation efficiency. Particle size is relatively easy to adjust by controlling flow rates within microfluidic channels, with higher flow rates generally producing smaller particles. However, continuous tuning of the particle rigidity remains challenging. By manipulation of the interfacial water layer between hydrophobic and amphiphilic components during nanoparticle formation, future designs may achieve greater control over rigidity, which is critical for improving cellular uptake and biodistribution. While shape tuning using microfluidic chips has not yet been fully explored in biomedical applications, advances in materials science may enable this aspect in the future, offering further customization of the nanovesicle properties.The integration of nanovesicle assembly and surface modification within a single microfluidic platform presents challenges due to the differing speeds of these processes. Nanovesicle assembly is typically rapid, whereas surface modifications, such as those involving functional biomolecules, occur more slowly and often require purification steps. Recent advances, such as rotary valve designs and single-axis camshaft mechanisms, offer precise control over flow mixing at different stages of the process, allowing for the automation of nanovesicle assembly and surface modification, thereby improving batch-to-batch reproducibility.In conclusion, microfluidic technologies represent a promising approach for the development of multifunctional nanovesicles with the potential to address key challenges in drug delivery and precision medicine. While obstacles related to cytotoxicity, scalability, and reproducibility remain, innovations in chip design, materials, and automation are paving the way for broader application in clinical settings. Future research, potentially incorporating machine learning, could further optimize the relationship between nanovesicle properties and biological outcomes, advancing the use of microfluidic technologies for therapeutic delivery.