In humans, a collection of diverse proteins known as lectins are responsible for binding to glycan epitopes. Recognition of extracellular glycan epitopes represents a key facet of the innate immune response in eukaryotes. These epitopes can be used to distinguish cell types, self‐from non‐self, and perhaps even pathogenic from commensal bacteria. Understanding human lectin specificity can uncover mechanisms underlying innate immunity. Additionally, lectins represent attractive reagents for the development of glycan‐reading probes. Here, we demonstrate our efforts to develop and deploy a pipeline for recombinant expression, functionalization, and characterization of a subset of human lectins, including C‐type lectins, ficolins, jacalin‐type lectins, galectins, and intelectins. Target lectins can be efficiently expressed and purified, functionalized with either biotin or a fluorophore via sortase‐mediated ligation under flow, and screened for glycan binding. The success of this pipeline has been validated in preliminary studies showing that the recombinant lectins can engage with glycans from mammalian glycan arrays, bacterial glycan arrays, purified mucins, or human biological samples. These experiments highlight the potential of lectins as valuable molecular probes that fill a gap in the current molecular toolkit for glycan analysis.Support or Funding InformationNIH U01 CA231079‐01