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Abstract Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF‐SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two‐dimensional gel electrophoresis (2D SDS‐PAGE). In addition, a total of 8 HCP were identified using matrix‐assisted laser desorption/ionization‐mass spectroscopy (MALDI‐MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron‐sized particles which could lead to the plugging of the filters.