With the advantages of rapid analysis, high sensitivity, and multicomponent identification, mass spectrometry (MS) is recognized as an appealing choice for high-throughput screening (HTS) analysis. Aiming at the small size, simple operation, and adequate performance, the development of miniature mass spectrometers has made great progress over the last 2 decades. Besides the essential analytical performance, simple operation and HTS capability are two other crucial features desired in miniature MS instruments. In this paper, an induced selfaspiration vacuum electrospray ionization source (ISA-VESI) was developed and coupled to a miniature ion trap mass spectrometer. A special timing sequence was designed to synchronize all the operation steps in each measurement, including dual-pulse sample injection, multipulse gas injection, MS analysis, and the movement of the homemade HTS platform used as the sampler. Then, the automatic high-throughput analysis of multiple samples can be accomplished with close coordination among the sample delivery, the sample introduction and ionization, and the ion trap operation. The measurement time of each ISA-VESI-MS analysis was about 7 s, with a sample consumption of less than 100 nL.