Herbicides containing glyphosate undergo decomposition mainly by microorganisms. Monitoring this process requires an application of simple, quick and cheap instrumental methods. Thin layer chromatography was used in the present work to determine organic compounds in fermentation broth and absorption UV-Vis spectroscopy to determine orthophosphate ions (Pi). The obtained chromatograms of fermentation broth showed glyphosate biodegradation, but not with the use of commonly known route with the formation of glycin, aminomethylphosphonic acid (AMPA) and orthophosphate ions (Pi). This fact was confirmed by spectroscopic studies used for the parallel determination of concentrations of inorganic phosphorus (Pi), the levels of which were very low.