In-depth proteome profiling still relies widely on extensive off-line peptide fractionation to reduce the complexity of the proteome. However, the fractionation of protein samples in the sub-microgram range can be limited owing to sample loss during the fractionation process. Thus, it is essential to develop efficient microscale fractionation strategies. Here, we describe a modified method for high-pH peptide fractionation using C18-StageTips. Compared to a conventional strategy, our method generates improved peptide and protein identification rates under the same LC-MS/MS conditions. Finally, proteome profiling of sub-regions of the mouse brain suggested that our method can be used to analyze the proteome in-depth with limited amounts of protein samples.