Abstract A colorimetric assay has been developed for quantitative analysis of active biomass of Fusarium fujikuroi , based on the reduction of the tetrazolium salt 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) when menadione was present as an electron coupling agent. The optimum assay-conditions were set as 200 μg/ml XTT, 5 μM menadione and one-hour reaction time. Under these settings, the produced formazan displayed a linear relationship with F. fujikuroi biomass. This method was subsequently applied to evaluate the cell growth behavior, which showed a positive correlation with the carbon source consumption and gibberellin biosynthesis under the industrial fermentation conditions. Our results showed that the XTT-menadione assay is a valuable tool in analyzing the industrial fermentation process of F. fujikuroi , especially when the medium contains insoluble and complex components. Graphical abstract Unlabelled Image Highlights • The selected assay conditions are 200 μg/ml XTT, 5 μM menadione and 1 h reaction time. • The produced formazan showed in a linear relationship with the fungal biomass. • The biomass behavior showed a positive correlation with the glucose level. • The cells secreted GA3 in the stationary phase, especially the late stationary phase. [ABSTRACT FROM AUTHOR]