In biopharmaceutical production, ultrafiltration and diafiltration (UF/DF) becomes essential when high concentrations and tight control over the final formulation of drug substance is needed. Currently, the possibilities for direct inline monitoring of this process step are limited. This study evaluates the effectiveness of three spectroscopic techniques, i.e., mid-infrared (MIR), Raman, and variable pathlength ultraviolet (UV), or inline monitoring of protein concentration. Results demonstrate that all three techniques are suitable for monitoring this operation, with root mean squared errors of prediction (RMSEP) of 5.0, 3.2, and 3.2% for MIR, variable pathlength UV, and Raman spectroscopy, respectively.