Recent advancements in plastic and reconstructive surgery have harnessed the therapeutic potential of enzymatically isolated adipose-derived regenerative cells (ADRCs) as a minimally invasive and effective solution for tissue regeneration. Isolated from the stromal vascular fraction (SVF) of adipose tissue, ADRCs comprise a heterogeneous population of cells, including adipose-derived stem cells, endothelial cells, pericytes, and macrophages, each contributing uniquely to the regenerative cascade. Nonenzymatic methods aiming at extracting the SVF from adipose tissue result mainly in small tissue fragments that also contain adipocytes. As a result, these methods do not result in final cell suspensions that only contain SVF cells. In contrast, enzymatic methods enable to isolate the SVF cells from lipoaspirate without the presence of adipocytes and tissue fragments in the final cell suspension, resulting in high cell counts while maintaining cell viability and functionality. This chapter provides an overview of the enzymatic isolation protocols and evaluates the cellular composition, viability, and clinical applicability of ADRCs in reconstructive procedures. By examining the role of enzymatically isolated ADRCs in promoting tissue repair, angiogenesis, and immunomodulation, this chapter underscores the transformative impact of these cells on the field of regenerative medicine. Furthermore, a short summary of clinical outcomes of application of ADRCs in soft tissue augmentation, chronic wound healing, and musculoskeletal tissue repair is provided, demonstrating the capacity of these cells to effectively support the body’s intrinsic healing processes. Given the rising demand for autologous point-of-care treatments, therapies based on the injection of ADRCs stand at the forefront of innovation in reconstructive surgery, offering promising, safe, and effective treatment options for patients.