In vivo electroporation (IVE) has gained significant attention as an efficient, versatile, and minimally invasive technique for intracellular delivery of nucleic acids, proteins, and other biologically active molecules. By applying electrical pulses to transiently permeabilize cell membranes, this technique enhances the uptake and expression of therapeutic agents, enabling diverse applications in gene therapy, cancer immunotherapy, vaccination, tissue regeneration, and genome editing. This review summarizes recent advances in electroporation technology, including innovations in device design, optimization of pulse parameters, and combinatorial strategies, with a focus on translational biomedical research. Key challenges such as tissue response heterogeneity, safety concerns, and reproducibility are also discussed. By examining ongoing clinical trials and future directions, we aim to offer insights into addressing these barriers and accelerating the clinical translation of in vivo electroporation for therapeutic use.