Intracellular/extracellular free ions, such as Ca2+, H+ and Mg2+ concentrations are important for vital cell functions, such as excitability, membrane potential, cell volume regulation, cell proliferation and apoptosis. Under physiological conditions, intracellular and extracellular free ion concentrations are fine controlled; however, they are dysregulated in a number of diseases. Utilizing digital cell imaging, high throughput screening (HTS) and ion sensitive electrodes, we offer measurements of intracellular and extracellular free ion, such as Ca2+, H+ and Mg2+concentrations to test effects of lead compounds on these free ions, and further elucidate the mechanisms underlying action of the compounds on the ions.