<P>The development of real-time monitoring and an improved control system was performed according to a process analytical technology initiative focusing on the benefits of the pharmaceutical industry. As an extension of the results of pioneering Raman-based closed-loop feedback control of crystallization and synthesis, a bioprocess, namely, the enzymatic hydrolysis of lactose, was investigated in this work. For this purpose, an effective control system was developed to maintain the lactose concentration at a level of 20 g/L. Real-time evaluation of Raman spectra was accomplished using classical least-squares (CLS) and partial least-squares (PLS) multivariate data analysis methods. Formation of oligosaccharides during the hydrolysis of lactose causes inaccuracy in the CLS evaluation, and thus, optimization of lactose hydrolysis was needed to decrease oligosaccharide formation. After optimization, control of lactose hydrolysis could be achieved. To improve real-time evaluation, the PLS method was used, resulting in better evaluation of spectra during control experiments.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/oprdfk/2016/oprdfk.2016.20.issue-10/acs.oprd.6b00212/production/images/medium/op-2016-00212z_0011.gif'></P>