In bottom-up proteomics, the complexity of the proteome requires advanced peptide separation and/or fractionation methods to acquire an in-depth understanding of protein profiles. Proposed earlier as a solution-phase ion manipulation device, liquid phase ion traps (LPITs) were used in front of mass spectrometers to accumulate target ions for improved detection sensitivity. In this work, an LPIT-reversed phase liquid chromatography-tandem mass spectrometry (LPIT-RPLC-MS/MS) platform was established for deep bottom-up proteomics. LPIT was used here as a robust and effective method for peptide fractionation, which also shows good reproducibility and sensitivity on both qualitative and quantitative levels. LPIT separates peptides based on their effective charges and hydrodynamic radii, which is orthogonal to that of RPLC. With excellent orthogonality, the integration of LPIT with RPLC-MS/MS could effectively increase the number of peptides and proteins being detected. When HeLa cells were analyzed, peptide and protein coverages were increased by ∼89.2% and 50.3%, respectively. With high efficiency and low cost, this LPIT-based peptide fraction method could potentially be used in routine deep bottom-up proteomics.