In negative chromatography, the impurities bind on the adsorbent, and the product is allowed to flow through the chromatographic column. Negative chromatography is an alternative to positive chromatography under certain circumstances and has been used to purify various biomolecules. For this review, a detailed survey of the performance of reported studies on negative chromatography was conducted. The applications of negative chromatography in the capture and intermediate purification steps for biomolecules (e.g., plasmid DNA, antibodies, enzymes, hemoglobin, virus particles and cells) are reviewed. The negative chromatographic adsorbents adsorb the impurities through surface charge, hydrophobic interaction at specific sites on the surface, hydrophobic interaction, hydrogen bonding and functional groups. Examples of applications of negative chromatography according to the type of chromatography matrix used are summarized and discussed. In addition, the effects of operating conditions (initial protein concentration, buffer ions, pH and salt concentration) are discussed, and the criteria for choosing negative or positive chromatography are summarized. The literature survey showed that there will be future limitations and challenges ahead in implementation of negative chromatography. Possible solutions to the limitations and challenges of negative chromatography and future trends for developing negative chromatography are discussed.