Background: The homology of aldosterone- and cortisol-producing enzymes, aldosterone synthase (CYP11B2) and 11β-hydroxylase, complicates identification of selective CYP11B2 inhibitors required for antihypertensive treatment or for imaging approaches in patients with primary aldosteronism. To improve preclinical evaluation of novel CYP11B2-targeting compounds, we developed a flow-through cell culture system that provides insights into kinetics of steroidogenesis and inhibitory responses at CYP11B2 active sites. Methods: NCI-H295RA and HAC-15 cells were cultured in ibidi flow chambers under constant culture medium flow. Supernatants were collected hourly before and after treatment with (R)-1-[1-(4-iodophenyl)ethyl]-1H-imidazole-5-carboxylic acid azetidinylamide (IMAZA), a non-selective CYP11B1/B2 inhibitor, or the potential CYP11B2 inhibitors ID-69 and ID-191. Steroid profiles were analyzed by liquid chromatography-tandem mass spectrometry. Steady state approximation in steroidogenesis allowed mathematical modeling-based calculation of metabolic fluxes and relative rate constants of biocatalytic steps. Results: An optimized flow-through system is now available to characterize inhibitory responses at the three catalytic sites of CYP11B2 in two steroid-producing cell lines. IMAZA non-selectively inhibited CYP11B 11β-hydroxylase function, while ID-69 and ID-191 affected the CYP11B2-specific 18-hydroxylase active site with minor effects on catalytic activity of 11β-hydroxylase. ID-191 simultaneously impaired catalytic activity of cortisol production, whereas ID-69 was highly selective for CYP11B2 inhibition. Conclusion: Our flow-through system provides insights into inhibitor-induced alterations of metabolic fluxes and enzymatic rate constants, and thus represents an improved preclinical model sytem for complex characterization of CYP11B2 inhibitors.