Knowledge of the speciation of heavy metals and the role of dissolved organic matter (DOM) in soil solution is a key to understand metal mobility and ecotoxicity. In this study, soil column-Donnan membrane technique (SC-DMT) was used to measure metal speciation of Cd, Cu, Ni, Pb, and Zn in eighteen soil solutions, covering a wide range of metal sources and concentrations. DOM in these soil solutions was also fractionated into humic acid (HA), fulvic acid (FA), hydrophilic acid (Hy), and hydrophobic neutral organic matter (HON) by a rapid batch technique using DAX-8 resin. Our results show that in soil solution Pb and Cu are dominant in complex form, whereas Cd, Ni and Zn mainly exist as free ions; for the whole range of soil solutions, only 26.2% of DOM is humic substances and consists mainly of fulvic acid (FA). The metal speciation measured by SC-DMT was compared to the predicted ones obtained via the NICA-Donnan model using the measured FA concentrations. The free ion concentrations predicted by speciation modeling were in good agreement with the SC-DMT measurement. Moreover, we show that to make accurate modeling of metal speciation in soil solutions, the knowledge of DOM composition, especially FA fraction, is the crucial information, especially for Cu and Cd; like in previous studies the modeling of Pb speciation is not optimal and an update of Pb generic binding parameters is required to reduce model prediction uncertainties.