Electrospray ionization (ESI) efficiency is known to be affected by the properties of the analytes, source design and source parameters. In this study, the ionization efficiency of 17 acidic compounds at various conditions in ESI negative ion mode was evaluated. Namely, the influence of organic solvent content in the mobile phase, ionization source parameters, ionization source geometry and functionality (conventional ESI, ESI with thermal focussing and with additional internal nebulizer gas) was studied. It was observed that the ionization efficiency in thermal focussing ESI is only marginally affected by the organic solvent composition, while for conventional ESI and ESI with internal nebulizer gas, the ionization efficiency increases significantly with increasing organic modifier content. For all ionization sources and mobile phase compositions, the ionization efficiency values between different setups showed good correlation. Copyright © 2016 John Wiley & Sons, Ltd.