The protein barrier-to-autointegration factor (BAF) and its interaction partners, the LEM (LAP2B, emerin, MAN1)-domain proteins, constitute a powerful cytoplasmic DNA defense mechanism. Invading DNA molecules are quickly bound by the BAF system and trapped in membrane compartments. This decreases the nuclear uptake of DNA from the cytoplasm. Inhibition of the BAF system is therefore expected to enhance the efficacy of non-viral DNA transfection agents. In this study, we introduced a protocol for the recombinant expression of soluble BAF and developed two ELISA-type assays to discover small molecule inhibitors of BAF-dependent DNA retention by high throughput screening (HTS). The proton pump inhibitor rabeprazole as well as three compounds of the Maybridge library were identified as inhibitors of the LEM-BAF-DNA interaction chain. The inhibition was based on adduct formation with BAF cysteine residues. An enhancing effect of the compounds on cell culture transfection, however, was not observed, which may be attributed to the reducing environment of the cytoplasm that prevents the adduct formation with BAF cysteine residues. The novel assays developed here can provide new tools to further study the biological functions of the BAF system, and may lead to the identification of suitable BAF inhibitors in future HTS campaigns.