The field of proteomics has emerged as a valuable analytical tool for elucidating cellular and biological systems at the molecular level. As there is an ever-growing demand for new highly automated, high-throughput, and sensitive analytical tools, a key challenge is the characterization of low abundance proteins that are crucial in modulating biological functions of cells and may also be associated with a number of diseases. If the detection modes are mainly dominated by fluorescence spectroscopy and mass spectrometry, the recent advances in microfluidic techniques have the potential to meet the requirements for sample treatment and processing. Microfluidic devices can address the future analytical needs of increased throughput, lower sample and reagent consumption, smaller size, and lower operating costs. It has been found that microfluidic-based enzymatic reactors can carry out protein digestion with high efficiency to facilitate subsequent reliable protein identification by peptide mass fingerprinting. Compared to conventional digestion approaches, microfluidic enzymatic reactors can not only accelerate digestion rate but also reduce enzyme autolysis and, ultimately, achieve the purpose of repetitive utilization.