In the homo-VHL system, the method's limitation in distinguishing between binary and ternary binding may mask potency differences, diminishing the contrast between AS2 and CM11 (Figure ). Consequently, it becomes challenging to differentiate the potency of CM11 and AS2, although both compounds display a similar trend in VHL engagement. In contrast, within the CRBN/VHL system, compounds 14a and AS4 demonstrated 4-fold and 2-fold lower potency, respectively, in live-cell assays (IC50live = 1.33 µM and 0.49 µM, Figure ) compared to permeabilised-cell assays (IC50perm = 0.30 µM and 0.22 µM). This suggests that limited cellular permeability restricts VHL target engagement for both compounds. Notably, the smaller disparity in binding affinity between live-cell and permeabilised-cell conditions for AS4 indicates that this compound possesses superior cell permeability relative to 14a, as reflected by an AI that is two-fold lower. Similarly, when tested for VHL engagement, both MZ1 and AS6 showed full engagement in cell-permeabilised mode (IC50perm = 0.53 µM and 0.17 µM, respectively). However, the most significant difference was observed in live-cell mode, where AS6 exhibited an IC50 only 10-fold lower than the permeabilised mode (IC50live = 1.22 µM), in contrast to the larger IC50 difference observed with MZ1 (20-fold, IC50live= 10.3 µM). Overall, FerroTACs demonstrated better permeability compared to the reference compounds, as indicated by the smaller differences in VHL engagement between permeabilised and live-cell modes. This ratio is depicted in Figure , where the AI values of FerroTACs are positioned closer to the diagonal corresponding to AI = 1, indicating maximal permeability. However, exceptions such as CM11/AS2 may highlight an inherent limitation of the assay.