Cell heterogeneity is widely present in the biological world and exists even in an isogenic population. Resolving the protein heterogeneity at the single cell level is of enormous biological and clinical relevance. However, single cell protein analysis has proven to be challenging due to extremely low amount of protein in a single cell and the huge complexity of proteome. This requires appropriate sampling and sensitive detection techniques. Here, a new approach, microfluidics combined with MALDI-TOF mass spectrometry was brought forward, for the analysis of proteins in single cells. The detection sensitivity of peptides as low as 300 molecules and of proteins as low as 10^6 molecules has been demonstrated. Furthermore, an immunoassay was successfully integrated in the microfluidic device for capturing the proteins of interest and further identifying them by subsequent enzymatic digestion. Moreover, an improved microfluidic platform was designed with separate chambers and valves, allowing the absolute quantification by employing iTRAQ tags or an isotopically labeled peptide. The study was further extended to analyze a protein in MCF-7 cell lysate. The approach capable of identifying and quantifying protein molecules in MCF-7 cells is promising for future proteomic studies at the single cell level.