There are several methods to detect phytotoxic effects of metabolites from plants. Generally, organic compounds are pre-dissolved in organic solvents and then in water to bioassay its activity on target plants. However, compounds solubility and pH in water after removing solvents are not totally controlled. Other bioassays are performed using DMSO and MES as a solvent and buffer medium to improve compounds solubility and stabilize pH respectively. The aim of this work was to compare different methods to assess phytotoxic effects of chemical compounds extracted from flowers of Acacia dealbata. Chemicals of fresh flowers were extracted in methanol for ten weeks and sequentially fractionated with hexane, ethyl acetate and water. The dry organic fractions were re-dissolved in its own solvents plus water and in DMSO buffered with MES. The phytotoxic effect of each fraction was bioassayed at 0, 5, 10, 25 and 50 ppm on the germination and seedling growth of Lactuca sativa. In general, the growth of L. sativa was significantly stimulated when acacia chemicals were pre-dissolved with DMSO. However, L. sativa growth was solvent, concentration and pH dependent when organic solvents were used. Plant germination was not affected in any case. Our results show that the phytotoxic effect of chemicals from A. dealbata flowers may be influenced by the dissolving procedure. DMSO-or organic solvent-based bioassays must be carefully selected depending on research interests. DMSO may be useful to find the full effect of isolated compounds and organic solvents may be used under an ecological approach.