Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein-ligand interactions. Currently, there is a growing need for breakthroughs in the application of HDX-MS analysis to protein-ligand interactions in highly complex biological samples such as cell lysates. However, HDX-MS analysis in such systems suffers from extreme spectral complexity as a result of high sample complexity and limited LC separation power due to the traditional use of short LC gradients. Here, we introduced protein thermal depletion (PTD) to reduce protein complexity in E. coli cell lysate for our subzero-temperature long gradient UPLC-HDX-MS platform (PTD-HDX-MS) to facilitate high-throughput analysis of protein-ligand interactions in cell lysates. We spiked bovine carbonic anhydrase II (CaII) and its inhibitor acetazolamide (AZM) into E. coli cell lysate as a model system in our study. We demonstrated that PTD at 60 °C greatly reduces protein complexity in cell lysates, while the AZM-targeted CaII complex remains in solution due to improved thermal stability upon binding. Using both PTD to reduce sample complexity and subzero-temperature long gradient UPLC to boost LC separation power, we successfully elucidated the interaction sites between AZM and CaII in E. coli cell lysate from the high-throughput HDX-MS analysis of thousands of deuterated peptides from hundreds of proteins. Our results highlight the great promise of the PTD-HDX-MS platform for the identification of ligand targets and characterization of protein-ligand interactions in highly complex biological samples such as cell lysates.