The drug discovery process increasingly relies on high-throughput sample analysis to accelerate the identification of viable drug candidates. Recently, chromatographic-free highthroughput mass spectrometry (HT-MS) technologies have emerged, significantly increasing sample readout speed and enabling the analysis of large sample sets. These HT-MS platforms continuously acquire data from various samples into a single data file, presenting challenges in applying distinctive data acquisition methods to specific samples. This study introduces a novel approach that integrates realtime sample loading status to activate sample-specific MS/MS data acquisition methods on the high-throughput acoustic ejection mass spectrometry platform. Effective method switching and high signal reproducibility were demonstrated across different data acquisition window durations in multiple reaction monitoring (MRM), high-resolution MRM (MRM HR ), and information-dependent acquisition modes. This advancement provides a user-friendly and robust solution to the method-setting challenges of HT-MS, expanding the implementation of HT-MS platforms in drug discovery and other high-throughput analytical applications.T he drug discovery process relies heavily on automated and integrated high-throughput sample analysis. While traditional methods like colorimetric microplate-based readers are prevalent, they often face limitations such as a restricted linear dynamic range and the necessity for label attachment schemes, which can alter equilibrium and kinetic analyses. In contrast, mass spectrometry (MS) coupled with liquid chromatography (LC) offers label-free, universal mass detection with high sensitivity, selectivity, and specificity. 1 However, the widespread adoption of LC-MS in highthroughput settings has been impeded by its relatively low analysis throughput, primarily due to the time required for complete elution from chromatographic columns. 2 In recent years, the emergence of chromatographic-free high-throughput mass spectrometry (HT-MS) technologies has expanded the utilization of MS in large sample set analysis. 3 These platforms, including matrix-assisted laser desorption ionization (MALDI), 4 laser diode thermal desorption (LDTD), 5 direct analysis in real-time (DART), 6 desorption electrospray ionization (DESI), 7,8 matrix-assisted laser desorption electrospray ionization (MALDESI), 9 liquid atmospheric pressure (LAP)-MALDI, 10 acoustic mist ionization (AMI), 11 and acoustic ejection mass spectrometry (AEMS), 12,13 have demonstrated significant improvements in sample readout speed, exceeding 10-fold compared to LC-MS, enabling the analysis of over 50 000 samples per day.