Tracheal reconstruction is indicated in cases of malignancy, traumatic injury, and subglottic or tracheal stenosis. Recent progress in airway transplantation has provided renewed optimism for potential solutions for defects involving more than half of the tracheal length in adults or one-third of the tracheal length in children. Biologic scaffolds derived from decellularized tissues and organs have shown great promise in tracheal allotransplantation, and cyclical decellularization techniques have been hypothesized as abrogating the need for immunosuppressive therapy. In this study, we performed a direct comparison of three decellularization protocols (Protocols A, B, and C) previously described in the literature, two of which were described in tracheal tissue (Protocols A and B). We concentrated on the immunogenicity within the epithelium and mucosa, quantified and qualified the extracellular matrix (ECM) components, and performed compliance measurements on large circumferential decellularized tracheal scaffolds following cyclical decellularization techniques using all three protocols. Quantitative measurements of glycosaminoglycans (GAGs) showed a significant decrease in the mucosal component following 17 cycles of all 3 protocols as well as a significant decrease of GAGs in the cartilaginous component following cycles 1, 9, and 17 of Protocol A and cycle 17 of Protocol C. Compliance measurements were also shown to be different between the protocols, with grafts becoming more compliant at physiologic pressures after cyclical decellularization with Protocols A and B and slightly less compliant but remaining similar to native trachea using Protocol C. Positive staining for anti-major histocompatibility complex Class I (anti-MHCI) and anti-MHCII remained within the submucosal glandular components despite multiple cycles of decellularization using all three protocols. This study illustrated that there are significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the decellularization protocol. Protocol B was shown to maintain the GAGs components despite an increase in tracheal compliance, while Protocol C decreases GAGs components following multiple cycles, despite showing a tracheal compliance resembling that of the native trachea at physiologic airway pressures.