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Of the many metabolites involved in any clinical condition, only a narrow range of biomarkers is currently being used in the clinical setting. A key to personalized medicine would be to extend this range. Metabolic fingerprinting provides a more comprehensive insight, but many methods used for metabolomics analysis are too complex and time-consuming to be diagnostically useful. Here, a rapid evaporative ionization mass spectrometry (REIMS) system for direct ex vivo real-time analysis of biofluids with minor sample pretreatment is detailed. The REIMS can be linked to various laser wavelength systems (such as optical parametric oscillator or CO 2 laser) and with automation for high-throughput analysis. Laser-induced sample evaporation occurs within seconds through radiative heating with the plume guided to the MS instrument. The presented procedure includes (i) laser setup with automation, (ii) analysis of biofluids (blood/urine/stool/saliva/sputum/breast milk) and (iii) data analysis. We provide the optimal settings for biofluid analysis and quality control, enabling sensitive, precise and robust analysis. Using the automated setup, 96 samples can be analyzed in ~35-40 min per ionization mode, with no intervention required. Metabolic fingerprints are made up of 2,000-4,000 features, for which relative quantification can be achieved at high repeatability when total ion current normalization is applied. With saliva and feces as example matrices, >70% of features had a coefficient of variance ≤30%. However, to achieve acceptable long-term reproducibility, additional normalizations by, e.g., LOESS are recommended, especially for positive ionization., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)