RATIONALE:Extractive electrospray ionization mass spectrometry (EESI-MS) was invented as a typical ambient mass spectrometry method (AMS) and has been used for analyzing complex liquid samples. Here, we designed a Venturi effect-based self-aspiration sampling device and applied it to the EESI-MS for high-throughput analysis of liquid sample. METHODS: A special concentric nebulizer was designed and employed to produce a suction force for the direct aspiration of liquid samples, followed by ionization and detection. This sample aspiration process was explained and optimized using computational fluid dynamics (CFD) analysis. Experiment data were recorded to exhibit the sensitivity, memory effect, inter-day reproducibility, throughput, and applicability of the self-aspiration sampling EESI-MS. RESULTS: The limit of detection (LOD) of this method was determined as 4.5 × 10 À10 g/mL (S/N = 3) for caffeine, and the sample throughput and relative standard deviation (RSD) for full scan mode can reach 0.67 samples/s and 4.76%, respectively. Even for MS/MS mode, the frequency can still be kept at 0.4 samples/s (RSD = 4.71%). Inter-day RSD examined in 1 week was below 10% for the signal of characteristic fragment ions of reserpine. Moreover, based on this method, the amount of caffeine in instant coffee was determined as 4.7%. This device was also proven to be suitable for the protein/peptide analysis. CONCLUSIONS: These experiment results, especially the amazing results on sample throughput and inter-day RSD, suggest that we provide a valuable device which can be used for the direct high-throughput qualitative/quantitative mass spectrometry analysis of real liquid samples in ambient. Copyright © 2016 John Wiley & Sons, Ltd.Ambient mass spectrometry (AMS) [1][2][3] has promoted the development of mass spectrometry (MS)-based high-throughput screening technology, [4][5][6] for its ability in rapid and direct analysis of various samples. Desorption electrospray ionization (DESI), [7][8][9][10] the first reported ambient ionization method, has been demonstrated in the high-throughput analysis of pharmaceutical samples, [11] immediately after its invention. During the same time period, direct analysis in real time (DART) [12][13][14][15] was invented, and several sample injection devices based on DART have been developed to increase the method's detection speed and throughput. [16,17] Both DESI and DART are mainly used to detect volatile, semi-volatile, or easily desorbed compounds from a solid surface, but they are inconvenient for liquid samples which need to be smeared on special surfaces.Extractive electrospray ionization (EESI) was invented originally for the direct detection of complex liquid samples [18,19] and has recently been successfully used in the analysis of exhaled gasses, [20,21] viscous samples, [22][23][24] solid surfaces, [25][26][27][28] etc. With an improved nanoEESI-MS method, Hu et al. [29] achieved the rapid analysis of a single sample within only 1.2 s. However, in nanoEESI-MS experiments, the solution ...