We developed, tested, and optimized two laboratory denitrification assays for both managers and scientists to assess the effects of xenobiotics on the denitrification process over 7 days (short batch assay, SBA) and 28 days (long semi-continuous assay, LSA). The assays facilitate (1) measuring the efficiency of the nitrate removal under the influence of xenobiotics, (2) determining the removal of the tested xenobiotic via adsorption or biotic decomposition, and (3) testing influencing parameters for optimizing the denitrification process. The adsorption of the xenobiotic was assessed by inhibiting all biological processes through the addition of HgCl2. Our tests demonstrate that the ratio of the initial nitrate concentration to the amount of bioavailable organic matter provided is essential to avoid organic carbon or nitrate limitation. While pH < 7 resulted in decreased denitrification, pH > 8 led to nitrite accumulation, indicating incomplete denitrification. Over durations of more than a week, weekly replenishments of the nitrate and the HgCl2, and weekly purging with argon gas to reduce oxygen concentrations are needed. The assays provide information about the accumulation of xenobiotics in the bioreactors necessary for an environmentally friendly treatment of the bioreactor fillings and provide insight into their potential to remove pesticides from polluted water resources.