Pyrylium salts are considered efficient chemical tags for amino groups. However, the apparent steric selectivity of pyrylium salts limits their application in the field of chemical labeling, especially during the labeling of sterically hindered compounds like amino acids, peptides, and proteins. Herein, we have investigated the effects of the α-substitution of pyrylium salts on their reactivity. We have also investigated the mechanism of nucleophilic reactions with pyrylium salts and further proposed that the reactivity of pyrylium salts mainly depends on the position and type of their substituents. A series of pyrylium salts were synthesized, and a highly active α-monosubstituted pyrylium salt, 2,4,5-triphenylpyrylium, was developed for efficient chemical labeling. All of the 15 amino acids studied were efficiently labeled under optimized reaction conditions. The 2,4,5-triphenylpyrylium salt was highly efficient in comparison to the previously reported 2,4,6-triphenylpyrylium salt developed for lysine-specific modifications. Furthermore, we successfully used 2,4,5-triphenylpyrylium salt for the hydrophobic labeling of peptides and protein hydrolysates. The most striking observation was that the ionization efficiency of short-chain multilabeled peptides in mixed samples, after derivatization, increased by up to 60 times. The increase in ionization efficiency gradually decreased with increasing peptide chain length. During the "soft" collision-induced dissociation (CID) process, the peptide was tagged at the N-terminus with 2,4,5-triphenylpyrylium, producing abundant a-type ions and b-type ions (Δ = 28), which eases the peptide resequencing process and assists in cracking the peptide codes. Moreover, 2,4,5-triphenylpyrylium has been utilized for the proteomic analysis of HeLa cell digests. In addition, 215 additional proteins were identified in the labeled products and the coverage of most proteins was improved.