<p>The paper deals with the study of the stability of solutions of 5-(2-aminoethyl)dithio-2-nitrobenzoate (ADNB), a chromogenic reagent for thiol detection, in a mixture with butyrylthiocholine iodide, a synthetic analog of the neurotransmitter acetylcholine. The aim of the study was to assess the suitability of these solutions for an enzymatic (cholinesterase-based) method for detecting cholinesterase inhibitors. The core of the study involved evaluating the thermal stability of the solutions at 60&#xa0;°C over a period of 150&#xa0;days. The results show that aqueous solutions of the ADNB-substrate mixture retained their original analytical properties after 30&#xa0;days of thermal stress, with some functionality still observed even after 90&#xa0;days. Similar results were obtained when 10% of the detergent slovasol or polyethylene glycol was added to the base solution to improve reagent solubility. The results were compared with an aqueous solution of the substrate and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent). Functionality was tested under conditions (enzyme acetylcholinesterase immobilized on a flat carrier) suitable for detecting the nerve agents VX and A-234 (novichok) at minimum quantities of &gt; 0.01&#xa0;µg and &lt; 0.01&#xa0;µg, respectively. The obtained data indicate a high stability of the studied solutions, enabling the development of new types of biosensors for a broad range of cholinesterase inhibitors.</p> Graphical abstract <p></p>