Key points Development of a flow cytometry (FCM) method for viability determination of S. acidocaldarius using the fluorescent dyes fluorescein diacetate and concanavalin A conjugated with rhodamine. Applicability of the developed method was shown via viability monitoring during a continuous cultivation with triggered pH shifts. A logarithmic trend was observed between the developed FCM method and the state-of-the-art method, plating assay.