For a comprehensive analysis of the mouse serum proteome, a customized mouse serum spectral library was constructed and utilized for quantitative analysis using DIA-MS. Our spectral library contained information on 1,106 mouse serum proteins. Creation of the spectral library was performed using Spectronaut Pulsar X by searching against the mouse protein sequences from the UniProtKB/Swiss-Prot database with the addition of a iRT fasta database.