Whole-cell patch-clamp-based electrophysiological techniques are powerful tools for examining the biophysical and pharmacological properties of ion channels. However, the recent validation of ion channels as novel drug targets necessitates the development of a faster screening method for ion channels. Therefore, we have developed a rapid, reliable, and sensitive cell-based high throughput screening (HTS) assay for T-type Ca2+ channels. We had previously constructed HEK293/alpha(1G)/Kir2.1 cell lines that stably expressed alpha(1G) and Kir2.1 subunits  and found that alpha(1G) T-type channel-sensitive Ca2+ signals were detected by the application of high concentrations of KCl under fura-2-based single cell measurements of intracellular Ca2+ concentration ([Ca2+](i)). In the present study, we applied HEK293/alpha (1G)/Kir2.1 cells to the FDSS6000 (Functional Drug Screening System) to develop a fast and reliable cell-based HTS method for alpha(1G) T-type Ca2+ channels. After detecting 70 mM KCl-induced [Ca2+](i) increases using the FDSS6000 system, we verified this new alpha(1G) channel HTS system by examining two T-type Ca2+ channel blockers, Ni2+ and mibefradil, and measuring the Z'-factor (Z' factor = 0.66) in 96-well plates. Furthermore, we assayed selected 3,4-dihydroquinazolin derivatives using this FDSS6000-based alpha(1G) channel HTS system at the level of IC(50) values and compared the results with those obtained from whole-cell patch-clamp recordings. Taken together, our results suggest that the FDSS6000-based alpha(1G) channel HTS system is a fast and feasible assay for alpha(1G) T-type Ca2+ channels. This assay can be utilized as a primary screening method for T-type Ca2+ channel-targeted chemicals and for the development of HTS systems for other types of ion channels.