Background The purpose of this work was to find a rapid method for glycerol detection during microbial fermentations. The method requirements were, first, to avoid sample pretreatment, and second, to measure glycerol precisely especially out of fermentation broth. Results This was achieved by combining two reaction principles - the Malaprade reaction and the Hantzsch reaction. In the Malaprade reaction, glycerol is converted into formaldehyde. This forms a dye in the Hantzsch reaction after which adsorption is than detected. The subsequent assay was investigated with two different fermentation media, a chemically undefined and a chemically defined media, used for Pichia pastoris fermentation. In both media, as well as in real fermentation samples, glycerol content could be reproducibly detected with the method. Moreover, measurements were more precise than using a standard glycerol detection kit. Conclusions With this rapid assay, glycerol could be detected easily in microbial fermentation broth. It is reliable over a wide concentration range including advantages such as an easy assay set-up, a short assay time and no sample pretreatment.