Update information. The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (AlkalinepH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH=8) and directly introduced to the mass spectrometer through nanoelectrospray ionization. The identified peptides overlapped between AlkalinepH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (LowpH-MS/MS) were as low as 45%, indicating that these two methods were complementary to each other. In addition, the AlkalinepH-MS/MS method showed identification capacity for a higher proportion of peptides with low molecular weight (MW), positive grand average of hydropathy (GRAVY), or high isoelectric point (pI). Moreover, the AlkalinepH-MS/MS method had a bias toward histidine, lysine, methionine, and proline- containing peptides. The complementarity of AlkalinepH-MS/MS and LowpH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating of 60-min AlkalinepH-MS/MS and 60-min LowpH-MS/MS method outperformed the conventional 120-min LowpH-MS/MS method in the identification of amino acid variants and protein groups. Our AlkalinepH-MS/MS method was an excellent complementary and alternative method to the LowpH-MS/MS method for global proteomic analysis.