This work focuses on investigating the fractionation of proteins by membrane ultrafiltration under controlled permeate flux. The results obtained using beta-lactoglobulin and gamma-globulin lead to the conclusion that under controlled flux it is possible to reduce transmission of the oversize protein and thus improve protein fractionation. This technique ensures the highest possible rejection for the most rejected component, which is crucial to obtain a good protein separation. To monitor transmission of each protein a fluorescence detection technique was developed, using different fluorescent probes to label different proteins. This allows off-line and continuous on-line monitoring of protein transmission. Direct visualisation of protein rejection by ultrafiltration under different operating conditions was also possible via fluorescence microscopy using fluorescent probes.