The relatively new high-resolution MRM (MRMHR) approach with the Triple-TOF MS running under nanoflow was adopted in this study to solidify further the findings of SWATH-MS results of differentially expressed proteins found across the time points in the chick vitreous for the benefit of low peptide amount consumption. Twelve up-regulated and 26 down-regulated proteins were found across all time points compared to day 7 using SWATH-MS. Several DEPs, such as alpha-fetoprotein, cadherins family group, neurocan, and reelin involved in structural and growth-related pathways, were validated for the first time using MRMHR under this experimental condition.