Humic acids as a water soluble fraction of humus play important role in the plant growth. Even though structure of humic acids is not clear, their spectroscopic properties can reveal us useful information. To investigate influence of humic acids reorganization, HPSEC fractionation at mild fractionation conditions has been carried out. Reconstruction of secondary chromatograms of separated fractions showed molecular size increase and increased response on fluorescence detector, although neither molecular size change nor significant absorbance increase was observed on UV-VIS detector. However, bigger aggregates, that didn't fluoresce in the unfractionated sample started to fluoresce. The reason for this behavior is not some change of spectral properties of a specific fraction, but a general fluorescence increase indicating humic acid reorganization. Therefore spectroscopic changes can be used as a tool for monitoring their reorganization, which might play an important role in nutrients soil plant mobility and should be studied in more details from that perspective.