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Rapid Evaporative Ionisation Mass Spectrometry (REIMS) has been shown to quickly and accurately speciate microorganisms based upon their species-specific lipid profile. Previous work by members of this group showed that the use of a handheld bipolar probe allowed REIMS to analyse microbial cultures directly from culture plates, without any prior preparation. However, this method of analysis would likely be unsuitable for a high-throughput clinical microbiology laboratory. Here, we report on the creation of a customised platform which enables automated, high-throughput REIMS analysis, which requires minimal user input and operation; and suitable for use in clinical microbiology laboratories. The ability of this high-throughput platform to speciate clinically important microorganisms was tested through the analysis of 375 different clinical isolates, collected from distinct patient samples, from 25 microbial species. After optimisation of our data analysis approach, we achieved substantially similar results between the two REIMS approaches. For handheld bipolar probe REIMS a speciation accuracy of 96.3% was achieved, whilst for high-throughput REIMS, an accuracy of 93.9% was achieved. Thus, high-throughput REIMS offers an alternative mass spectrometry based method for the rapid and accurate identification of clinically important microorganisms in clinical laboratories without any pre-analysis preparative steps.