Proper functioning wound healing strategies are sparse. Adequate vascular formation to the injured area, as well as replacement of the volume loss, is fundamental in soft tissue repair. Tissue engineering strategies have been proposed for the treatment of these injury sites. Novel cell-free substance, human adipose tissue extract (ATE), has been previously shown to induce in vitro angiogenesis and adipogenesis and in vivo soft tissue formation. This study reports the translation of ATE preparation from laboratory to the operating room (OR). ATE samples for this study were derived from adipose tissue obtained with the water-jet assisted liposuction technique from 27 healthy patients. The variables studied included incubation time (15, 30, and 45 min), temperature (room temperature vs. 37°C), and filter type to determine the optimal method yielding the most consistent total protein content, as well as consistent and high expression of adipose-derived growth factors and cytokines, including: vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, leptin, and insulin-like growth factor. Following the optimization, samples were produced in the OR and tested for their sterility. No significant differences were observed when comparing extract incubation time points or incubation temperature. Nonetheless, when studying the different filter types used, a syringe filter with PES membrane with larger filter area showed significantly higher protein concentration (p ≤ 0.018). When studying the different growth factor concentrations, ELISA results showed less variation in cytokine concentrations in the OR samples with the optimized protocol. All of the OR samples were tested sterile. The devised protocol is an easy and reproducible OR-ready method for ATE generation. As an attractive source of growth factors, ATE is a promising alternative in the vast field of tissue engineering. Its clinical applications include volume replacement as a complement to fillers and improvement of the permanence of fat grafts and wound healing, among other bioactive functions.