We anticipated that a key feature of the PhABit platform would be the opportunity to perform follow-up studies to further characterise the hits and enable appropriate triaging. First, concentration-response studies were performed to measure PhABit binding affinity. Hit PhABits were incubated at a range of concentrations with BRD4-BD1 (1 µM) before irradiation and analysis by LC-MS. In all cases, a concentration-dependent reduction in the crosslinking yield was observed (Figure ). The resulting curves could be fit to calculate pKD values, and enabled deconvolution of the crosslinking event; the gradient of the curve is determined by the non-covalent KD of the fragment, while the plateau represents the maximum crosslinking efficiency of the PhABit (Figure ). Importantly, this maximum is independent of the potency of the fragment, and only measures crosslinking efficiency versus other carbene quenching processes. Thus, concentration-response studies enabled differentiation of weak binders with high crosslinking efficiency (e.g. 1) and potent binders with poor crosslinking efficiency (e.g. 3). To validate the PhABit-generated pKD values, the fragments were also screened by SPR (pKD) and TR-FRET (pIC50) assay, which showed good correlation (SPR R 2 =0.82 and TR-FRET R 2 =0.90, Figure ). The concentration-response study thus offers an expedient method for determining the potency of hit PhABits.