Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells. The competitive binding model enabled the determination of common CA inhibitors’ dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.