Abstract One of the methods by which large and non-volatile molecules, especially those of biochemical interest, can be made amenable to mass spectrometry (MS) is electrospray (ES) ionisation. In ES MS the technique of electrohydrodynamic spraying of liquids is used to produce gas-phase ions from sample molecules present in a diluted solution, which are subsequently transferred into a mass analyser. The special conditions required for effective ion formation (voltages, flow rates, solution parameters such as conductivity, sample concentration, solvent composition) are described, as well as the instrumentation used to transfer and mass-analyse the gas-phase ions. Several variants of the ES MS technique are presented which have been developed to meet certain analytical requirements (such as spraying of highly conductive or aqueous solutions, coupling to separation techniques as liquid chromatography or capillary zone electrophoresis, or the analysis of very small sample quantities). The mechanisms of liquid charging, aerosol formation and ion release from the charged droplets (as far as they are understood) are briefly discussed, and the question is addressed in how far the ions observed in the ES mass spectrum reflect the state of the sample molecules in solution. Some examples of applications from the field of peptide/protein analysis are given, covering molecular weight determination, sequence and spatial structure determination, and studies on non-covalent molecular interactions. Finally, a short comment is made on the limitations of ES MS and, from a practitoner’s point of view, which problems need still to be solved.