The focus of this Ph.D. thesis is the theoretical, computational, and experimental analysis of electrohydrodynamics and ionization in the Array of Micromachined UltraSonic Electrospray (AMUSE) ion source. The AMUSE ion source, for mass spectrometry (MS), is a mechanically-driven, droplet-based ion source that can independently control charge separation and droplet formation, thereby conceptually differing from electrospray ionization (ESI). This aspect allows for low voltage soft ionization of a variety of analytes and flexibility in the choice of solvents, providing a multifunctional interface between liquid chromatography and mass spectrometry for bioanalysis. AMUSE is a versatile device that operates in an array format, enabling a wide range of configurations, including high-throughput and multiplexed modes of operation. This thesis establishes an in-depth understanding of the fundamental physics of analyte charging and electrokinetic charge separation in order to enhance droplet charging and ionization efficiency. A detailed electrohydrodynamic (EHD) computational model of charge transport during the droplet formation cycle in the AMUSE ion source is developed, coupling fluid dynamics, pressure and electric fields, and charge transport in multiphase flow. The developed EHD model presents a powerful tool for optimal design and operation of the AMUSE ion source, providing insight into the microscopic details of physicochemical phenomena, on the microsecond time scale. Analyte charging and electrohydrodynamics in AMUSE are characterized using dynamic charge generation measurements and high-spatial-resolution stroboscopic visualization of ejection phenomena. Specific regimes of charge transport, which control the final droplet charging, have been identified through experimental characterization and simulations. A scale analysis of the ejection phenomena provides a parametric regime map for AMUSE ejection modes in the presence of an external electric field. This analysis identifies the transition between inertia-dominated (mechanical) and electrically-dominated (electrospraying) ejection, where inertial and electric forces are comparable, producing coupled electromechanical atomization. The understanding of analyte charging and charge separation developed through complimentary theoretical and experimental investigations is utilized to improve signal abundance, sensitivity, and stability of the AMUSE-MS response. Finally, these tools and fundamental understanding provide a sound groundwork for the optimization of the AMUSE ion source and future MS investigations.