Acid-solubilized collagen type I (COL1) can form highly organized, three-dimensional scaffolds for a wide variety of bioengineering and cell culture applications. A rapid COL1 isolation method would be a valuable tool for both basic and translational researchers because conventional techniques require days or weeks to complete, typically use nonhuman animal tissues as a source material, and do not efficiently purify autologous COL1 from small samples. Here, we describe a 3-h method to isolate COL1 from rabbit, lamb, and human skin in sufficient quantities for fabrication of autologously derived tissues by using a rapid agitation technique and incorporating centrifugal filtration units into a traditional isolation procedure. We demonstrate that the purified product is comparable to traditional preparations using polyacrylamide gel electrophoresis, transmission electron microscopy, and collagen content assays. In addition, our COL1 is able to support myogenic cell growth and direct orientation of these cells in vitro. Importantly, this ultrarapid COL1 isolation procedure increases the feasibility of autologous COL1 use in humans as well as overall safety for clinical patients.