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Abstract Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.