Adjuvants are commonly used to improve the efficacy of pesticides, both as part of formulations or as a separate addition to the spray tank. One such adjuvant is an ethoxylated soybean oil (ESBO). As the chemical composition of ESBO and the fractions mainly responsible for the adjuvant-effect are unknown, the aim of the present study was to elucidate the composition of ESBO applying preparative high-performance liquid chromatography (prep. HPLC) and to evaluate the adjuvant-effect of isolated fractions in comparison to the commercial product. By prep. HPLC, four main fractions could be determined and isolated. Abutilon theophrasti (velvetleaf) and Zea mays (maize) were used as test species to evaluate the adjuvant effect of isolated fractions or ESBO on the herbicidal efficacy of bromoxynil octanoate, foramsulfuron & iodosulfuron, and technical grade bromoxynil potassium. Experiments were conducted under greenhouse conditions and dry weight of leaves, leaf area, and quantum yield of photosystem II were evaluated as response parameters. Leaf area and dry weight responses showed that there was a tendency towards late eluting (non-polar) fractions enhancing the efficacy of hydrophilic herbicides to a higher extent than earlier eluting (polar) fractions. For example, foramsulfuron & iodosulfuron in combination with the most non-polar fraction resulted in a 61% lower dry weight of velvetleaf compared with the herbicide treatment without adjuvant. The analysis of quantum yield resulted in the lowest values for the treatment with bromoxynil octanoate (BO) mixed with ESBO and foramsulfuron & iodosulfuron (FI) combined with the most polar fraction, respectively. The detection of an active compound within an adjuvant could be a new direction for additive optimisation and, hence, very precise recommendations for herbicide-adjuvant mixtures might be deduced.Abbrevations: AI = active ingredient, BO = bromoxynil octanoate, BP = bromoxynil potassium, ChF = chlorophyll Į fluorescence, DAA = days after application, DW = dry weight, ESBO = ethoxylated soybean oil, FI = foramsulfuron & iodosulfuron, LA = leaf area, prep. HPLC = preparative high-performance liquid chromatography, PS II = photosystem II, Y(II) = quantum yield of photosystem II