ABSTRACT A standard protocol to evaluate the effects of biostimulants on plant physiology is still lacking. The proton pumps present in the vacuolar and plasma membranes are the primary agents responsible for the regulation of the electrochemical gradient that energizes the nutrient uptake system and acid growth mechanism of plant cells. In this study, two of these enzymes were characterized as biochemical markers of biostimulant activity. A simple and fast protocol based on the degree of root acidification using a pH sensitive dye and the Micro-Tom tomato as a plant model is proposed as an efficient methodology to prove the efficacy of biostimulants that are claimed to improve nutrient acquisition and root growth. The results agree with the data from more conventional, expensive and time-consuming proton pump assays. A direct correlation was found between plasmalemma proton-adenosine triphosphatase (H+-ATPase) activation and the amount of rhizosphere acidification observed in the bromocresol gel. Moreover, roots of the diageotropica (dgt) Micro-Tom plants, defective in auxin responses, barely acidify bromocresol purple gel even in the presence of indole-3-acetic acid (IAA, 1 μM). The biostimulant TEA (vermicompost water extract, 25 %) enhances proton extrusion by 40 % in wild type (WT) plants, but no effect was induced in dgt plants. These results reinforce the notion that the class of biostimulant known as humic substances stimulates plant proton pumps and promotes root growth by exerting an auxin-like bioactivity and establish the usefulness of an economically and technically feasible assay to certify this kind of biostimulant.