Abstract: Mass spectrometry (MS) is now established as a technique that can screen compounds at a throughput that is comparable to fluorescent-based techniques with fewer false positives and without the need for labeled probes. Our group and others are now exploring other types of assays that can benefit from ultra-high-throughput mass spectrometry (uHT-MS). This presentation will show progress being made in analyzing proteins and processing this data to aid in covalent and nuisance compound screens. Also, examples of how uHT-MS is being used to monitor reactions products when coupled to high-throughput experimentation reaction condition screens will be given. In a recent cellular assay to find glutaminase inhibitors, other endogenous metabolites besides glutamine and glutamic acid were found to have relative concentrations changed upon compound treatment. Based on these findings, we hypothesized that we could use signal changes from uHT-MS to establish a metabolic phenotypic profiling screen. Initial studies towards developing this profiling scheme will be presented. Included will be experimental data and processes to establish controls, methods and informatics capabilities required to perform these metabolomic phenotypic profiling studies. Using UMAP and unsupervised clustering, endogenous metabolites and unidentified mass features detected by the mass spectrometer can be clustered based on cell type or by compound treatment. Moreover, using this workflow we have reinvestigated false positive glutaminase cellular screening hits that were not binders to glutaminase and found differences in their metabolite profiles compared to confirmed glutaminase inhibitors. This suggests that high throughput metabolomic profiling could quickly differentiate true hits from false positives without the need for lower throughput biophysical binding assays.