Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated 'MDTCS' variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues are the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope have preponderance compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest activity reductions were observed in the most autoantibody-resistant variants (15-35% residual activity in FRETS-VWF73). Among these, a triple-alanine mutant RARAA showed activity in a VWF multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from iTTP patients, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.