LC-ESI/MS/MS-based shotgun proteomics is currently the most commonly used approach for the identification and quantification of proteins in large-scale studies of biomarker discovery. In the past several years, the shotgun proteomics technologies have been refined toward further enhancement of proteome coverage. In the complex series of protocols involved in shotgun proteomics, however, loss of proteolytic peptides during the lyophilization step prior to the LC/MS/MS injection has been relatively neglected despite the fact that the dissolution of the hydrophobic peptides in lyophilized samples is difficult in 0.05–0.1% TFA or formic acid, causing substantial loss of precious peptide samples. In order to prevent the loss of peptide samples during this step, we devised a new protocol using Invitrosol (IVS), a commercially available surfactant compatible with ESI-MS; by dissolving the lyophilized peptides in IVS, we show improved recovery of hydrophobic peptides, leading to enhanced coverage of proteome. Thus, the use of IVS in the recovery step of lyophilized peptides will help the shotgun proteomics analysis by expanding the proteome coverage, which would significantly promote the discovery and development of new diagnostic markers and therapeutic targets.