A novel in situ magnetic separator is presented in which the separation unit is directly mounted on the bioreactor. Therefore, it is possible to collect the magnetic particles directly from the reaction liquid and transfer them in one step sterile out of the reactor. By consecutive flushing, the particles are concentrated and separated from the rest. The operating parameters were determined and evaluated experimentally and by computational fluid dynamics. The separator was tested in combination with a 5-L bioreactor with glass flask. Hereby, the accumulation kinetics of the particles were analyzed and used for a simple theoretical model.