The preparation and optimization of a new monolithic chymotrypsin bioreactor for online protein digestion is described. Silica monolithic supports have been activated with epoxide functionalities following an optimized in situ procedure and used for covalent immobilization of chymotrypsin in one-step reaction under different conditions. A total of four bioreactors were prepared and characterized in terms of the amount of immobilized enzyme and apparent active units by using a standard substrate, N-benzoyl-L-tyrosine p-nitroanilide (BTPNA). The stability of the bioreactors was evaluated and the morphology of the support after immobilization and use was studied by SEM analysis. The proteolytic activity of the optimized chymotrypsin bioreactor was evaluated using HSA as a model protein by online coupling of the bioreactor with LC-ESI-MS. With the online protocol, complete protein digestion in 120 min was achieved with a sequence coverage of 97.3%.