Traditionally most downstream bioprocesses have been operated without real-time knowledge of product and key contaminants, yielding little confidence in their operation and the impact on subsequent operations. A rapid UV-vis spectral prediction technique has been successfully demonstrated for the at-line characterization of a large scale continuous flocculation process in terms of RNA, key protein contaminants, and cell debris. A comparison was made between the spectral predictions and retrospective wet chemical assays, and a highly linear correlation was obtained. The spectral analysis technique allowed for real-time system information, which was applied to control the flocculation process to maintain satisfactory process performance, even when subjected to given possible process disturbances.