Continuous flow infrared matrix-assisted laser desorption electrospray ionization (CF IR MALDESI) mass spectrometry was demonstrated for the on-line analysis of liquid samples. Samples in aqueous solution were flowed through a 50 µm i.d. fused-silica capillary at a flow rate of 1–6 µL/min. As analyte aqueous solution flowed through the capillary, a liquid sample bead formed at the capillary tip. A pulsed infrared optical parametric oscillator (OPO) laser with wavelength of 2.94 µm and a 20 Hz repetition rate was focused onto the capillary tip for sample desorption and ablation. The plume of ejected sample was entrained in an electrospray to form ions by MALDESI. The resulting ions were sampled into an ion trap mass spectrometer for analysis. Using CF IR MALDESI, several chemical and biochemical reactions were monitored on-line: the chelation of 1,10-phenanthroline with iron(II), insulin denaturation with 1,4-dithiothreitol, and tryptic digestion of cytochrome c. Copyright © 2010 John Wiley & Sons, Ltd.