The advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolite concentrations, it does not consider the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework, Network Response Analysis (NRA), for rational genetic strain design. NRA is cast as a Mixed-Integer Linear Programming problem that integrates MCA, Thermodynamically-based Flux Analysis (TFA), biologically relevant constraints, as well as genome editing restrictions into a comprehensive platform for identifying metabolic engineering targets. We show that the NRA formulation and its core constraints are equivalent to the ones of Flux Balance Analysis (FBA) and TFA, which allows it to be used for a wide range of optimization criteria and with various physiological constraints. We also show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.