Herein, a rapid electrochemical screening of yeasts (Saccharomyces cerevisiae) in vitro mode depending on their optical density, cultivation time and growth medium used was conducted in 3 min by palladium nanoparticles (Pd-NPs)-modified electrodes. Pd-NPs-modified electrodes operated in cyclic voltammetry mode at low scan rates, i.e. 5–20 mV/s supported a low oxidative process in the yeast extracellular matrix. The electrochemical screening relied on an efficient electrooxidation of secondary metabolites, i.e. organohydrazines formed in the extracellular medium as a result of microbial activity of yeast cells. More importantly, during the study the impact of fundamental parameters, viz. type of the matrix and pH on electroanalytical response of Pd-NPs-based electrodes in real fermentation medium was investigated in detail. The efficiency of the proposed in vitro electrochemical screening of yeast extracellular matrix was not affected by pH of the samples or composition of the multicomponent medium, but more likely exclusively depended on the presence of organohydrazines. The potential of this electroanalytical approach towards profiling of the extracellular matrix of Saccharomyces cerevisiae was compared with results obtained by gas chromatography mass-spectrometry (GC–MS) and genetically encoded biosensor (ro-GFP2) assays.