During cell cultivation processes for the production of biopharmaceuticals, good process performance and good product quality can be ensured by online monitoring of critical process parameters (e.g. temperature, pH, or dissolved oxygen). These data can be used in real-time for process control, as suggested by the process analytical technology (PAT) initiative. Today, solutions for real-time monitoring of parameters such as concentrations of cells, main nutrients, and metabolism by-products are developing, but applications of these more complex tools in industrial settings are still limited. Here, we evaluated the use of dielectric spectroscopy (DS) and near-infrared spectroscopy (NIRS) as PAT tools for a perfusion PER.C6® cultivation process. We showed that DS enabled predictions of viable cell density from the cultivation vessel, with a root mean square error of prediction (RMSEP) of 4.4% of the calibration range. Additionally, predictions of glucose and lactate concentrations from the cultivation vessel (RMSEP of 10 and 14%, respectively) and from the perfusion stream (RMSEP of 12 and 10%, respectively) were achieved with NIRS. We also showed that the perfusion stream offers great opportunities for noninvasive, yet frequent process monitoring. Accurate online monitoring of critical process parameters with PAT tools is the essential first step toward increased control of process output.