Recently, we have developed probe electrospray ionization (PESI) that uses a solid needle. In this system, the probe needle moves up and down along the vertical axis by a motor-driven system. At the highest position of the probe needle, electrospray is generated by applying a high voltage. In this study, we applied PESI directly to biological samples such as urine, mouse brain, mouse liver, salmon egg, and fruits (orange, banana, etc.). Strong ion signals for almost all the samples were obtained. The amount of liquid sample picked up by the needle is as small as pL or less, making PESI a promising non-invasive technique for detecting biomolecules in living systems such as cells. Therefore, PESI may be useful as a versatile and ready-to-use semi-online analytical tool in the fields of medicine, pharmaceuticals, agriculture, food science, etc. Copyright © 2008 John Wiley & Sons, Ltd.