This review discusses the questions if emergence of antibiotic resistance may be affected by the application of different methods for quantification of mutant frequencies and if so far disregarded physiologically relevant conditions at the foci of infection should be considered and if novel analytical methods should be applied. Regulatory authorities are currently requesting information about resistance development following exposure of pathogens either to constant or fluctuating antibiotic concentrations. This optional application of two different methods implies that data should be comparable but they differed by up to eight orders of magnitude when using one method or the other. Mutant-frequencies were low following exposure to constant concentrations but high following exposure to fluctuating concentrations. Furthermore, mutant frequencies differed significantly if bacteria were either routinely grown in commercial media with low mutant frequencies or, as it would be pathophysiologically more relevant, in humanized/human media with high mutant frequencies. Thus, results generated by using conventional methods may be inconsistent or may even represent artefacts thus causing misinterpretations. Information about modes of action and mechanisms of resistance is requested based on the assumption that target interactions would be identical under different experimental conditions. However, targets being essential under routine conditions get lost under pathophysiologically relevant conditions. Also, the limitation to evaluate cross-resistances within the class only, provided an agent of an existing class is being developed, is just one-sided. Subtle structural homologies between different drug classes and natural compounds are sufficient to select for cross-resistance. Furthermore, cross-resistance emerged due to downstream effects triggered by structurally unrelated antibiotics finally leading into common networks of signal transduction pathways. Therefore, emergence of resistance should not only be addressed from a target-oriented position but also from a physiological perspective linked to human and bacterial physiology, regulatory networks, and from a biochemical perspective considering structural homologies of antibiotics and stressors.