Background: ASCC are rare radiosensitive cancers commonly associated with human papillomavirus infection (HPV). Improvements in patient outcomes have been relatively inert in the last 4 decades with 5 year OS reported between 60-70%. This is compounded by the fact that there is a paucity of preclinical ASCC models to study tumour biology and assess novel therapeutics. Organoid technology is a powerful translational tool to personalise therapy and predict patient’s response to treatment. To date, there is no published literature on ASCC organoid models. We set out to establish and characterise a novel panel of ASCC patient derived tumouroids. Methods: Prospective patients were recruited to the Anal Tissue Bank after informed consent at Peter MacCallum Cancer Centre. Patient information and clinical data were extracted from electronic medical records to correlate with laboratory results. Fresh tumour tissues were collected and processed at the laboratory. Results: 7 robust long term ASCC organoid models were cultivated from a varied disease spectrum including primary, local persistent/recurrent and metastatic disease. They have been validated from their tissue of origin and display similar characteristics to their parent tumour biopsies by histology, immunohistochemistry, RNA, whole exome sequencing and HPV profiling. The ASCC organoids display heterogeneity to standard of care treatment including 5 fluorouracil, Mitomycin C, radiotherapy and targeted PIK3CA and CDKN2A inhibitor therapies. Conclusion: This panel of patient derived ASCC organoids accurately recapitulate parent tumour molecular and genetic characteristics. This is an invaluable resource for disease modelling and a useful platform for novel therapy discovery.