Mixed-mode chromatography is a chromatographic method that utilizes more than one form of interactions between the stationary phase and the solutes in a feed stream. Compared with other types of chromatography, mixed-mode chromatography is advantageous in its salt-independent adsorption, facile elution by charge repulsion, and unique selectivity. Hence, it has already proved beneficial for the separation of proteins as well as other purposes. In this article, mixed-mode ligands for protein purification have been reviewed. These ligands usually have an aliphatic or aromatic group as the hydrophobic moiety and an amino, carboxyl or sulfonic group as the ionic moiety. Heterocyclic groups are good ligand candidates for their unique hydrophobicity and dissociation property. Hydrogen bonding groups also have influences on the performance of mixed-mode adsorbents. These principles should be considered in the screening and design of mixed-mode ligands. Strategies for the design of synthetic affinity ligands, especially the bioinformatics and combinatorial methods, may be adopted for mixed-mode ligand design. More efforts are needed for the development of rational design and screening methods for mixed-mode protein ligands by sophisticated computational and experimental approaches.