PTEN is an important tumor suppressor protein that is regulated by ubiquitination events which are modulated by deubiquitinases, or enzymes that remove ubiquitin from substrate proteins. As ubiquitinated substrates are beneficial to study deubiquitinase activity and substrate recognition, we have previously developed a semisynthetic strategy to site-specifically install a monoubiquitin on PTEN. This strategy uses a non-natural aminoAla-Cys functionality as a convenient alternative to the synthetically more challenging natural isopeptide linkage. However, the effective processing of this linkage by deubiquitinases other than by the deubiquitinase USP7 has not been evaluated. Therefore, we assessed whether the aminoAla-Cys linked monoubiquitinated PTEN can be processed by other known deubiquitinases. We found that USP10, USP11, and USP15 processed monoubiquitinated PTEN but BAP1 and OTUD3 could not under the conditions tested. This study demonstrates that ubiquitin linked to the aminoAla-Cys functionality is hydrolyzable by members of the USP family deubiquitinases and enables the systematic evaluation of deubiquitinase activities toward monoubiquitinated protein substrates.