<p>Humans are exposed to a multitude of substances at the same time; thus, toxicity assessment for mixtures of several substances and not only for the individual substances is critical. An important challenge in testing the mixture effects of many substances in vitro is the variability between individual experiments, also named ‘day-to-day variability’. Two steps, each consisting of independent experiments, are needed when mixtures are tested. In the first experimental step, for each substance, the concentration–response relationship is usually measured with 6–10 concentrations for at least 3 experiments, and EC₂₀ values, i.e., the concentration where a fitted curve intersects with a viability value of 80%, are determined. These values are used as reference values and in the second experimental step, the mixture of several substances is based on these individual EC₂₀ values. Since the mixture experiments are new, independent experiments, typically conducted on different days, day-to-day variability can lead to deviations in the cytotoxicity at the reference EC₂₀ concentration, i.e., to higher or lower observed viabilities. In this work, a procedure is proposed, how a single concentration per substance tested in the same experiment as the mixture can be used to reliably adjust for day-to-day variability. In the here-established procedure, a specific additive model denoted as ‘budget approach’ is introduced as a reference model to explore potential positive or negative interaction effects between substances.</p>