The main protease (3CL M pro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target . To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 M pro . To aid rational drug design, we determined a neutron structure of M pro in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature . We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free M pro, revealing modulation of the active-site protonation states upon telaprevir binding . We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the M pro active site . Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 M pro.