Literature on decellularization of human auricular cartilage is currently in-sufficient. Utomo et al.15 have provided a protocol however it didn’t specify the number of decellularization cycles required to produce an optimized scaffold. Similarly in the study by Gong et al.16, they have not clarified details of experimental steps. In addition the cartilage is of a porcine source only. This study is the first to compare multiple protocols for decellularization of human auricular cartilage with the detergent/enzymatic method proposed in protocol B having shown to have been the most effective. The steps for protocols A to C were chosen based on the success of methods from previous study groups. The process of freeze thaw has shown to accelerate decellularization23 when applied to different tissues and this can minimize the duration of exposure to chemical forms. Adding a wet freeze thaw cycle in PBS allowed for cellular remnants to be washed away from the scaffold after thawing at room temperature as well as exerting greater mechanical pressure on the cartilage in physically decellularizing it. Detergent/enzymatic methods can damage the structure of the ECM if prolonged22 and so physical techniques can shorten the exposure time. The use of EDTA in protocol C was adapted from Utomo et al.15 who had used it successfully in their decelullarization process. In addition the use of DNAse as well as SDC amongst the different protocols has been reported to effectively decellularize cartilage in the literature17. Trypsin was used in protocol B as it has shown to accelerate decellularization in the case of hyaline cartilage of the trachea as reported by Gomez et al.18.