Electrospray ionization (ESI) ion trap mass spectrometers with relatively low resolution are frequently used for the analysis of natural products and peptides. Although ESI spectra of multiply charged protein molecules also can be measured on this type of devices, only average spectra are produced for the majority of naturally occurring proteins. Evaluating such ESI protein spectra would provide valuable information about the native state of investigated proteins. However, no suitable and freely available software could be found which allows the charge state determination and molecular weight calculation of single proteins from average ESI-MS data. Therefore, an algorithm based on standard deviation optimization (scatter minimization) was implemented for the analysis of protein ESI-MS data. The resulting software ESIprot was tested with ESI-MS data of six intact reference proteins between 12.4 and 66.7 kDa. In all cases, the correct charge states could be determined. The obtained absolute mass errors were in a range between −0.2 and 1.2 Da, the relative errors below 30 ppm. The possible mass accuracy allows for valid conclusions about the actual condition of proteins. Moreover, the ESIprot algorithm demonstrates an extraordinary robustness and allows spectral interpretation from as little as two peaks, given sufficient quality of the provided m/z data, without the necessity for peak intensity data. ESIprot is independent from the raw data format and the computer platform, making it a versatile tool for mass spectrometrists. The program code was released under the open-source GPLv3 license to support future developments of mass spectrometry software. Copyright © 2010 John Wiley & Sons, Ltd.