Affinity purification is one of the most powerful separation techniques extensively employed both at laboratory and production scales. While antibodies still represent the gold standard affinity reagents, others derived from non-immunoglobulin scaffolds emerged as interesting alternatives in particular for affinity purification. The lower costs of production, fast ligand development, and high robustness are appealing advantages of non-immunoglobulin scaffolds. These have successfully been used in the affinity purification of relevant targets as antibodies, human serum albumin, transferrin, and other biomarkers, as reviewed in this work. Furthermore, a critical assessment on the strengths, weaknesses, opportunities, and threats related with the implementation of non-immunoglobulin scaffolds as ligands in affinity purification are discussed. Biotechnol. Bioeng. 2017;114: 481-491. (c) 2016 Wiley Periodicals, Inc.