The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological, Chemical and Environmental sciences (IIBR) and is being evaluated. Development of an efficient downstream purification process (DSP) enables advancing the vaccine to clinical trials. The DSP must eliminate impurities, either process- or product -related, to yield sufficient product with high purity, potency, and quality. To acquire critical information on process restrictions and qualities, incorporation of on-line monitoring is vital. Application of on-line monitoring should significantly impact process yield, product quality and economy of the entire process. Here, we describe an on-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining concentrations of metabolites, nutrients and a host cell protein (HCP) by the automatic chemistry analyzer Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adaptation of this technique during the purification process improves process yield, product quality and enhances the economy of the entire downstream process of biotechnology and bio pharmaceutical products.