<p>High-throughput screening of protein domains enables the systematic discovery of protein sequences that encode specific cellular functions. Fluorescence-activated cell sorting-based assays have long been the standard readout for such screens but remain time- and resource-intensive, imposing practical limits on library size and coverage. Here we describe a scalable magnetic separation-based workflow that provides an alternative to fluorescence-activated cell sorting for screening large protein libraries in mammalian cells. We engineered a modular synthetic surface marker, consisting of a fusion between the fragment crystallizable (Fc) region of human immunoglobulin G and the transmembrane domain of platelet-derived growth factor receptor-β, that allows cells to be magnetically separated on the basis of surface reporter expression using Protein G-coated magnetic beads. The procedure covers pooled library cloning, lentiviral delivery, magnetic separation and sequencing-based quantification, enabling reproducible screening of more than 100,000 protein domain variants. The approach is suitable for the identification of functional protein domains capable of transcriptional and post-transcriptional RNA regulation and may lead to the selection of improved transmembrane domains for efficient protein surface display. The entire workflow, from library design to data analysis, can be completed in 4–6 weeks and requires skills in cell culture, molecular cloning and computational techniques. This scalable and accessible Protocol enables researchers to systematically measure protein domain functions across biological contexts, thus accelerating both biological discovery and protein engineering.</p>