Preclinical assessment of drug-induced proarrhythmicity is typically evaluated by the potency of the drug to block the potassium human ether-à-go-go-related gene (hERG) channels, which is currently quantified by the IC₅₀. However, channel block depends on the experimental conditions. Our aim is to improve the evaluation of the blocking potency of drugs by designing experimental stimulation protocols to measure the IC₅₀ that will help to decide whether the IC₅₀ is representative enough. We used the state-of-the-art mathematical models of the cardiac electrophysiological activity to design three stimulation protocols that enhance the differences in the probabilities to occupy a certain conformational state of the channel and, therefore, the potential differences in the blocking effects of a compound. We simulated an extensive set of 144 in silico I_Kr blockers with different kinetics and affinities to conformational states of the channel and we also experimentally validated our key predictions. Our results show that the IC₅₀ protocol dependency relied on the tested compounds. Some of them showed no differences or small differences on the IC₅₀ value, which suggests that the IC₅₀ could be a good indicator of the blocking potency in these cases. However, others provided highly protocol dependent IC₅₀ values, which could differ by even 2 orders of magnitude. Moreover, the protocols yielding the maximum IC₅₀ and minimum IC₅₀ depended on the drug, which complicates the definition of a "standard" protocol to minimize the influence of the stimulation protocol on the IC₅₀ measurement in safety pharmacology. As a conclusion, we propose the adoption of our three-protocol IC₅₀ assay to estimate the potency to block hERG in vitro. If the IC₅₀ values obtained for a compound are similar, then the IC₅₀ could be used as an indicator of its blocking potency, otherwise kinetics and state-dependent binding properties should be accounted.