Fragment-based drug discovery is a powerful complement to conventional high-throughput screening, especially for difficult targets. Screening low-molecular-weight fragments usually requires highly sensitive biophysical methods, because of the generally low affinity of the identified ligands. Here, we developed a cell-based fragment screening assay (cellFy) that allows sensitive identification of fragment hits in a physiologically more relevant environment, in contrast to isolated target screenings in solution. For this, a fluorescently labeled multivalent reporter was employed, enabling direct measurement of displacement by low-molecular-weight fragments without requiring enzymatic reactions or receptor activation. We applied this technique to identify hits against two challenging targets of the C-type lectin receptor (CLR) family: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN) and Langerin. Both receptors are involved in pathogen recognition and initiation of an immune response, which renders them attractive targets for immune modulation. Because of their shallow and hydrophilic primary binding site, hit identification for CLRs is challenging and druglike ligands for CLRs are sparse. Screening of a fragment library followed by hit validation identified several promising candidates for further fragment evolution for DC-SIGN. In addition, a multiplexed assay format was developed for simultaneous screening against multiple CLRs, allowing a selectivity counterscreening. Overall, this sensitive cell-based fragment screening assay provides a powerful tool for rapid identification of bioactive fragments, even for difficult targets.