There is a clinical need to provide replacement tracheal tissue for the pediatric population affected by congenital defects, as current surgical solutions are not universally applicable. A potential solution is to use tissue engineered scaffold as the framework for regenerating autologous tissue. Rabbit trachea were used and different detergents (Triton x-100 and sodium deoxycholate) and enzymes (DNAse/RNAse) investigated to create a decellularization protocol. Each reagent was initially tested individually and the outcome used to design a combined protocol. At each stage the resultant scaffold was assessed histologically, molecularly for acellularity and matrix preservation. Immunogenicity of the final scaffold was assessed by implantation into a rat model for 4 weeks. Both enzymes and detergents were required to produce a completely acellular (DNA content 42.78 ng/mg) scaffold with preserved collagen and elastin however, GAG content were reduced (8.78 ± 1.35 vs. 5.5 ± 4.8). Following in vivo implantation the scaffold elicited minimal immune response and showed significant cellular infiltration and vasculogenesis. The luminal aspect of the implanted scaffold showed infiltration of host derived cells, which were positive for pan cytokeratin. It is possible to create biologically derived biocompatible scaffolds to address specific pediatric clinical problems. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2126-2135, 2017.