Affinity-based chromatography assays encompass the use of solid supports containing
immobilized biological targets to monitor binding events in the isolation, identification
and/or characterization of bioactive compounds. This powerful bioanalytical technique allows
the screening of potential binders through fast analyses that can be directly performed using
isolated substances or complex matrices. An overview of the recent researches in frontal and
zonal affinity-based chromatography screening assays, which has been used as a tool in the
identification and characterization of new anti-cancer agents, is discussed. In addition, a critical
evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed.