One of the most important challenges of drug development is identifying whether a candidate molecule can efficiently reach and engage intracellular targets. Cell-based assays are usually employed to address this issue; nevertheless, many compounds that appear promising in vitro fail in cellular or in vivo contexts due to low intracellular activity or off-target effects. In this work, we employed ligand-based in-cell 19F NMR to characterize ligand-target interactions in the cellular environment. We investigated the binding of newly synthesized fluorinated sulfonamide derivatives to various cytosolic isoforms of carbonic anhydrase in living human cells. We assessed target engagement and membrane permeability of each ligand and provided a qualitative affinity ranking of the compounds toward each isoform. The fluorinated compounds characterized in this study are promising scaffolds for the development of isoform-selective inhibitors and may serve as chemical probes for future competitive screenings of nonfluorinated drug candidates in cells.