Quantitative scanning transmission electron microscopy (STEM) is a powerful tool for the characterization of nano-materials. Absolute composition determination for ternary III–V semiconductors by direct comparison of experiment and simulation is well established. Here, we show a method to determine the composition of quaternary III–V semiconductors with two elements on each sub lattice from the intensities of one STEM image. As an example, this is applied to (GaIn)(AsBi). The feasibility of the method is shown in a simulation study that also explores the influence of detector angles and specimen thickness. Additionally, the method is applied to an experimental STEM image of a (GaIn)(AsBi) quantum well grown by metal organic vapour phase epitaxy. The obtained concentrations are in good agreement with X-ray diffraction and photoluminescence results.