Strategies for one‐pot peptide ligation enable chemists to access synthetic proteins at a high yield in a short time. Herein, we report a novel one‐pot multi‐segments ligation strategy using N‐terminal thiazolidine (Thz) peptide and a newly designed formaldehyde scavenger. Among the designed 2‐aminobenzamide‐based aldehyde scavengers, 2‐amino‐5‐methoxy‐N′,N′‐dimethylbenzohydrazide (AMDBH) can remarkably convert Thz into unprotected cysteine at pH 4.0. Furthermore, AMDBH degrades Thz at a considerably low rate at pH 7.5, and thioester degradation caused by this scavenger is negligible. As a result, we have developed an efficient one‐pot peptide ligation strategy by simply repetitively changing the pH with AMDBH. Finally, we synthesize mono‐ubiquitinated histone H2A.Z (209 amino acids) via AMDBH‐mediated one‐pot four‐segment peptide ligation in good yield.