In this study, we present the use of microreactors packed with immobilized trypsin particles for the rapid and efficient bottom-up analysis of proteins by on-line immobilized enzyme microreactor capillary electrophoresis mass spectrometry (IMER-CE-MS). The results obtained digesting β-lactoglogulin (β-LG) off-line with free trypsin in solution and with immobilized trypsin particles were taken as a reference for the optimization of the on-line protein digestion. Under the optimized conditions, on-line digestion, separation and characterization of the protein digests were possible in less than 30 min. The limit of detection for complete sequence coverage was around 10 µg mL-1 (~500 µM) of β-LG, the repeatability was comparable to the off-line digestion methods and the microreactor could be reused until thirty times. The good performance of IMER-CE-MS was also demonstrated for several other proteins as α-casein (α-CSN), β-casein (β-CSN), and κ-casein (κ-CSN), as well as for a complex protein mixture (an Escherichia coli whole cell lysate).