Although, with the live cell CETSA setup we could rank the prodrugs for overall potency, we cannot decouple the individual contributions of cellular permeability and prodrug unmasking rate. Towards understanding the rate of unmasking inside the live cells, we developed an in-cell time-resolved F-NMR to monitor the rate of POM group unmasking (Figure ). F-NMR is uniquely suited for this task due to the high receptivity of F, low background in biological samples and remarkable sensitivity of F chemical shifts to their chemical environment . The latter means that even the distant fluorine of MN551 could act as a reporter for the presence of the masking group on the phosphotyrosine moiety. The 19 F 1D NMR spectrum of a K562 cell suspension immediately after treatment with MN714 shows two broad, partially overlapping peaks 0.110 ppm apart (Figure ). While the downfield peak starts off with higher intensity, it loses intensity quickly and within an hour is overtaken by the growing upfield peak. Within 3 hours the downfield peak completely disappears and only the upfield peak is observed. To confirm that this time dependent change indeed corresponds to the unmasking of MN714, the cells were lysed after disappearance of the downfield peak and a 19 F 1D spectrum of the lysate collected. A single, sharp peak was observed that increased in intensity when the lysate was spiked with MN551 (Figure ). In contrast, spiking with MN714 gave rise to a new peak found 0.112 ppm downfield of the first peak, closely mirroring the peak differences observed in intact cells (Figure ). This suggests that the initially observed downfield peak is indeed caused by MN714, while the growing upfield peak corresponds to unmasked MN551. The partial overlap of the peaks and poor signal to noise prevent exact quantification of the unmasking kinetics, but the data confirms efficient unmasking of MN714 and suggests a half-life time of 1-1.5 hours.