Ultrafiltration (UF) is an effective technique for concentrating and fractionating proteins in liquid streams. Here we describe the determination of critical flux and limiting flux for ultrafiltration of protein solutions in a typical cross-flow filtration unit, also known as a tangential flow filtration (TFF) system, which can provide important information for developing fouling mitigation strategies in commercial operations. We also detail the protocols for acquiring the separation performance (e.g., flux, concentration factor, and selectivity) during the concentration and fractionation of proteins using UF membranes with desired molecular weight cutoff (MWCO). Typical procedures are also included for the clean-in-place (CIP) of the filtration unit.