High-resolution mass spectrometry has become a key technology in life sciences. Since it is often unfeasible to find pairs of analytes with an appropriate mass difference to actually quantify the resolution experimentally, resolution is usually calculated from the shape of a single mass peak. In this study we show that the commonly employed strategy yields a poor measure of true resolution since it does not account for interactions that take place between ions of very similar mass and might be further distorted by signal processing effects. We present a straightforward and easily adaptable method to create a ladder of mass pairs to experimentally quantify actual mass resolution over a wide m/z range, compare the experimental resolution to the single peak based calculated resolution, and demonstrate the applicability of mass resolution ladders to study interactions of similar ions in various types of widely used mass spectrometers.