Plasma membrane proteins’ profiling remains one of the toughest challenges for proteome researchers due to their hydrophobic nature and masked by the abundance of the peptides of cytoplasmic proteins. Traditional membrane fractionation with membrane organelle opening method have proven useful to spatially segregate and isolate hydrophobic membrane proteins. However, membrane fractionation has limitations such as contamination of cytoplasmic fractions and difficulty employing for cultured cells. In addition, the use of high amounts of detergent complicates the later steps of purification and affects MS compatibility. Chemical labeling methods especially Lysine reactive NHS ester containing chemical probes are popular in activity-based and membrane proteomics. Although the water-soluble probes are excellent for targeting the cell surface proteins, purification of labeled proteins are difficult and results in lower protein identification by 1D mass spectrometry. The aim of our study is to investigate the efficiency of biotinylated NHS ester probe in conjunction with carbonate wash treatment for improved purification plasma membrane and cell surface proteins in label free quantitative mass spectrometry. For this, we first investigated the efficiency of the cell surface biotinylation on live cultured HCT-116 cells and then combined the carbonate wash membrane prep method with the cell surface biotinylation to enrich plasma membrane proteins. With an introduction of on-bead digestion, this method has shown increased coverage and purity for enriching plasma membrane peptide (total 834 protein IDs) than the traditional fractionation cell surface biotinylation enrichment method (total 564 protein IDs) using 1D LC/MS/MS. The proteomics data was validated by Western blot and this method is employable for enriching both small (claudin-4) and large molecular weight membrane proteins (EGFR). This method is being tested for application on breast cancer cell and can be useful to track localized membrane proteomic change in stimulated carcinoma cell models.