While cell and gene therapies (C&GT) are gaining traction in the clinic with some notable successes, the manufacturing process for these therapies remains challenging and costly. The transient production of viral vectors used in C&GT, such as Adeno Associated Virus (AAV), is dependent on consistent upstream processes for batch-to-batch reproducibility. Such consistency is made possible with proper access to process parameters like viable and total cell densities. The  iLine F PRO (Ovizio) is an automated cell analyzer to monitor cellular dynamics through an on-line bioreactor connection. We evaluated the iLine F Pro as a tool for real-time HEK293 culture monitoring in manufacturing scale bioreactors for an AAV product. A good correlation between cell densities and viabilities was obtained with the iLine F PRO and the Vi-CELL XR for un-transfected and transfected cells. The developed algorithm can be used for new experiments to replicate the Vi-CELL counts in a label-free and on-line manner. The frequent measurements of the iLine F PRO (1 every hour) can be leveraged to predict when the target cell concentration for transfection will be reached. After transfection, a new cell population presenting morphological properties that could correspond to the transfected cell population was identified. Monitoring of this population would allow access to transfection efficiency in real time. In conclusion, we highlighted the high potential for on-line monitoring of the cell culture as a tool to increase process understanding, crucial during the development stage, and as a first step toward process automation.