Anticalin proteins are an emerging class of clinical-stage biopharmaceuticals with high potential as an alternative to antibodies. Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central β-barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop region results in Anticalin proteins that can recognize and tightly bind a wide range of medically relevant targets, from small molecules to peptides and proteins, as validated by X-ray structural analysis. Their robust format allows for modification in several ways, both as fusion proteins and by chemical conjugation, for example, to tune plasma half-life. Antagonistic Anticalin therapeutics have been developed for systemic administration (e.g., PRS-080: anti-hepcidin) or pulmonary delivery (e.g. PRS-060/AZD1402: anti-interleukin [IL]-4-Rα). Moreover, Anticalin proteins allow molecular formatting as bi- and even multispecific fusion proteins, especially in combination with antibodies that provide a second specificity. For example, PRS-343, which has recently entered clinical-stage development, combines an agonistic Anticalin targeting the costimulatory receptor 4-1BB with an antibody directed against the cancer antigen human epidermal growth factor receptor 2 (HER2), thus offering a novel treatment option in immuno-oncology.