The in vitro gas production technique was applied to evaluate the biological activity of tannins in sainfoin (Onobrychis viciifolia Scop.) hay cut at four progressive phenological stages. The buffalo (Bubalus bubalis) rumen fluid was used as inoculum, polyethylene glycol (PEG) was added as specific binding agent and an alfalfa hay (AH) was used as control. All samples were analysed for chemical composition and condensed tannin (CTs). According to the phenological stage of sainfoin, crude protein gradually decreased [from 219 to 122 g/kg dry matter (DM)] and neutral detergent fibre (NDF) increased (from 391 to 514 g/kg DM). Condensed tannins showed the highest value at the first phenological stage [31.5 mg catechin equivalents (CE)/g DM], and their level significantly decreased at the subsequent growth stages. Gas production and fermentation rate were significantly affected by substrate type and PEG addition. The first stage of sainfoin showed the lowest value of gas and volatile fatty acids production and the slowest fermentation kinetics compared with the other samples, when incubated without PEG, because of the CTs effect. The incubation with PEG improved the fermentation process of the first stage of sainfoin, but no effect was registered on AH, where CTs were not detected. Among the sainfoin samples, the slowest kinetics with PEG was observed in the hay harvested at the fourth stage, probably due to its chemical composition. The use of PEG allowed to verify the effect of tannins on the in vitro fermentation characteristics of sainfoin hay.