The aim of this study was to identify a protocol that could optimize the decellularization of human auricular cartilage. The technique was based on the success of previous methods applied in the case of tracheal17,18 and ear decellularization15. Our methodology consisted of applying three different decellularization protocols to human ear cartilage consisting of both physical and chemical methods. Physical methods of decellularization such as freeze thaw can help disrupt cell membranes19 as well as induce apoptosis of chondrocytes20. However the effect on degradation of the extracellular matrix structure has been proven to be insignificant. Szarko et al. reported no differences in glycosaminoglycan and collagen content after freeze thaw21 as well as reporting no change in the mean complex stiffness level. The process however initiates decellularization by inducing necrosis of cells whilst maintaining the three dimensional matrix structure. This reduces the duration of exposure to chemical methods of decellularization19. Chemical methods whilst effective can impair the mechanical properties of the matrix as Elder et al. noted decreased matrix strength when the exposure time of cartilage to SDS was increased from 2 hours to 8 hours22. Therefore a combination with physical decellularization techniques can help reduce the exposure time of cartilage to chemical methods. Freeze thaw action has been reported to accelerate the decellularization process in various tissues with lower nuclear content detected when combined with chemical techniques23. This study is the first to compare multiple decellularization protocols for human auricular cartilage and in doing so has identified a pathway for accelerating the process whilst preserving the structure of the native matrix.