Laser desorption/ionization mass spectrometry (LDI-MS) on porous silicon is a promising analytical strategy for the rapid detection of metabolites in biological matrices. We show that both oxidized and unoxidized porous silicon surfaces are useful in detecting protonated/deprotonated molecules from compounds when analyzed in mixtures. We demonstrate the feasibility of using this technique for the simultaneous detection of multiple analytes using a synthetic cocktail of 30 compounds commonly associated with prokaryotic and eukaryotic primary metabolism. The predominantly detected species were the protonated molecules or their sodium/potassium adducts in the positive-ion mode and the deprotonated molecules in the negative-ion mode, as opposed to fragments or other adducts. Surface oxidation appears to influence mass spectral responses; in particular, in the mixture we studied, the signal intensities of the hydrophobic amino acids were noticeably reduced. We show that whilst quantitative changes in individual analytes can be detected, ion suppression effects interfere when analyte levels are altered significantly. However, the response of most analytes was relatively unaffected by changes in the concentration of one of the analytes, so long as it was not allowed to dominate the mixture, which may limit the dynamic range of this approach. The differences in the response of the analytes when analyzed in mixtures could not be accounted for by considering their gas-phase and aqueous basicities alone. The implications of these findings in using the technique for metabolome analyses are discussed. Copyright © 2007 John Wiley & Sons, Ltd.