Phorbol ester analogs are a promising class of anticancer therapeutics and HIV latency reversing agents that interact with cellular membranes to recruit and activate protein kinase C (PKC) isoforms. However, it is unclear how these esters interact with membranes and how this might correlate with the biological activity of different phorbol ester analogs. Here, we have employed dynamic nuclear polarization (DNP) NMR to characterize phorbol esters in a native cellular context. The enhanced NMR sensitivity afforded by DNP and cryogenic operation reveals topological heterogeneity of 13C-21,22-phorbol-myristate-acetate (PMA) within T cells utilizing 13C-13C correlation and double quantum filtered NMR spectroscopy. We demonstrate the detection of therapeutically relevant amounts of PMA in T cells down to an upper limit of ∼60.0 pmol per million cells and identify PMA to be primarily localized in cellular membranes. Furthermore, we observe distinct 13C-21,22-PMA chemical shifts under DNP conditions in cells compared to model membrane samples and homogenized cell membranes, that cannot be accounted for by differences in conformation. We provide evidence for distinct membrane topologies of 13C-21,22-PMA in cell membranes that are consistent with shallow binding modes. This is the first of its kind in-cell DNP characterization of small molecules dissolved in the membranes of living cells, establishing in-cell DNP-NMR as an important method for the characterization of drug-membrane interactions within the context of the complex heterogeneous environment of intact cellular membranes. This work sets the stage for the identification of the in-cell structural interactions that govern the biological activity of phorbol esters.