<p>High-throughput chemical synthesis plays a critical role in generating compound libraries and optimizing reaction conditions. Increasing adoption of robots and multiwell formats means that hundreds of reactions can be accessed with ease. However, conventional methods for quantitative detection of the product (for example, by liquid chromatography–mass spectrometry) create a bottleneck in the workflow because analyses need to be customized separately for each sample. Here we describe a tandem mass spectrometry approach where the samples are analyzed directly using acoustic ejection mass spectrometry. This approach features simple method development enabling accurate quantification for reactions where a characteristic neutral lost fragment is common to both the chemical starting material and the expected reaction products. Combining this precise fragmentation signature with acoustic ejection mass spectrometry allows whole 384-well plates of chemical reaction data to be collected at the same pace as two liquid chromatography–mass spectrometry samples, while maintaining the same levels of accuracy. To explain the principles involved in designing these experiments, we show four examples of common medicinal chemistry transformations for C–N and C–C bond formation that have been used in the synthesis of analogs of cereblon binding molecular glues, antifungals, antibiotics, and building blocks for automated small molecule synthesis. The whole procedure requires ~2 days of work to complete, including the 384-well plate reaction setup, analytical sample preparation, mass spectrometry data collection and analysis.</p>