Circulating cholesterol is removed from blood plasma by cellular uptake via the low density lipoprotein receptor (LDLR). The inducible degrader of the LDLR (IDOL) is a post-translational regulator of the LDLR. IDOL homodimerization is essential for IDOL mediated degradation of the LDLR. Previous research utilised a genetic screening platform to screen for inhibitors of the IDOL homodimer PPI and identified cyclo-CFFLYT as an inhibitor of IDOL homodimerization. The affinity of the inhibitor was improved by incorporating non-natural phenylalanine analogues in place of the second phenylalanine, leading to the identification of cyclo-CF-Cha-LYT. This work aimed to investigate the structure-activity relationship of cyclo-CF-Cha-LYT and to optimise the SICLOPPS-RTHS (split-intein circular ligation of peptides and proteins – reverse twohybrid system) screening set-up to identify further inhibitors of IDOL. An alanine scan was performed by MST using Cy5 labelled YPet-IDOL, which identified cysteine and 3-cyclohexyl-Lalanine as critical residues of cyclo-CF-Cha-LYT. Chemically similar derivatives of cysteine were tested to investigate the interaction between the cysteine residue and IDOL. Only sulfurcontaining residues bound to IDOL which indicated that the residue was likely involved in a sulfurpi interaction, after eliminating disulfides and hydrogen bonding as possible interactions. CycloCF-Cha-LYT was not pursued any further due to a poor aqueous solubility, poor selectivity and a high clogP of 6.03. The SICLOPPS-RTHS screening set-up was adapted to be compatible with next-generation sequencing technologies. A screen was performed with the updated set-up and a CX5 SICLOPPS library, which identified two single-digit micromolar inhibitors of the IDOL homodimer, among other less active hits. The NGS screen was shown to be a success, as the hits obtained were more active and greater in number than those found in the traditional screen of the IDOL RTHS.