Natural organic matter (NOM) is present in the environment and could influence the migration of heavy metals/radionuclides. The dissolved fraction of NOM (DOM) is usually quantified using total organic carbon analysis or UV-visible spectrometry. Nonetheless, analysis using pattern recognition cannot provide the full spectrum of organic molecules contained in waters, especially low-molecular-weight compounds. In the context of nuclear performance assessment studies, ground waters may contain DOM and a key aspect is to quantify different categories of NOM types in order to further evaluate the transport and fate of radionuclides in the environment. Thus, a method for the quantification of DOM at the molecular level was developed, based on electrospray ionization mass spectrometry (ESI-MS). This method simultaneously gives structural information on DOM and the individual concentrations of these low-molecular-weight compounds without pretreatment and/or preconcentration of the samples. Several methods of quantification (internal calibration, calibrated addition of external standard, sequential tandem mass spectrometry) have been optimized and successfully applied to real natural samples. They are discussed in this paper with a focus on acidic compounds, which are the compounds that most probably could influence the migration of heavy metals and radionuclides in the clay rock pore water from the French Callovo-Oxfordian (COx) nuclear repository site. Quantification of in situ dissolved NOM from the COx has been performed using ESI-MS. For the first time to our knowledge, it was possible to give a quite exhaustive and quantitative inventory of the small organic compounds present without proceeding to any chemical treatment or sample crushing and for naturally occurring concentrations.