Sulfonamide antibiotics and Trimethoprim are antimicrobial substances used as growth promoters, for treatment and prophylactic issues in agriculture. These substances are found in animal sewage and ambient waters. Therefore it is likely that the substances can also be found in soil from fields where manure is applied. It is known that high concentrations of antimicrobial substances in an environmental compartment can cause a high selection pressure on microorganisms and result in more and more resistant bacteria to these substances. To understand more about the correlation of antimicrobial substances in the soil and resistant microorganism it is necessary to know what concentrations can be found in soil. To determine concentrations of these substances in soil an extraction method based on Accelerated Solvent Extraction (ASE) has been developed. The soil was extracted with a mixture of acetonitrile, acetone and water during 3 minutes at 80°C under a pressure of 100 bar. The volume of the soil extract was reduced to about 400 uL with N2-gas and than filtered. The filtered extract was diluted with 400 uL nano pure water and than analysed with HPLC-ESI-MS/MS. The absolute recoveries of this extraction method were as follows: Sulfamethazine 54%, Sulfadiazine 47%, Sulfathiazole 55%, Sulfamethoxazole 54%, Sulfadimethoxine 52%, Sulfachloropyridiazine 50% and Trimetoprim 40%. These values were all calculated using the isotope labeled internal standard with the exception of Trimetoprim and Sulfachloropyridiazine. The absolute recoveries were found to depend strongly on the contact time of the substances with the soil. Further more a very fast disappearance processe, which is not fully understood at the moment.