In eukaryotes, CTCF, a zinc finger DNA-binding protein, plays an important role in gene regulation through organising the three-dimensional architecture of the genome. Over the past two decades, there have been a large number of genomic studies investigating the role of CTCF in genome organisation of various cancer and normal tissues. However, the molecular mechanisms of CTCF function are not well understood. To understand the function of CTCF a map of interaction partners is needed. Here, we set up a two-step purification protocol coupled to liquid chromatography-tandem mass spectrometry to assess CTCF interaction partners in three different cancer cell lines, using label-free intensity-based absolute quantification (iBAQ). In addition, our interactome data suggest numerous proteins involved in gene regulation and chromatin remodeling as possible interaction partners of CTCF. To corroborate the high throughput interactome data, in vitro and cell-based pull-down experiments were performed.