3,5 - Dimethylpyrazolium glyceroborate is a nitrification inhibitor (a member of pyrazole derivatives) used for the fixation of nitrogen into the soil. In this study, an HPLC-DAD method was developed and validated for determination of 3,5 dimethylpyrazole in order to determine 3,5 - dimethylpyrazolium glyceroborate in fertilizer samples. For method development, analytical parameters like type of eluent solution and column filling material and device parameters like eluent flow rate, column oven temperature and measurement wavelength were all optimized. For method validation, implementations were performed for linearity, limit of detection (LOD), limit of quantification (LOQ), specificity, stability, intra- and inter -day precision and accuracy. The developed and validated method was used for inhibitor detection in nitrogenous fertilizers. Sample analyses were performed with 95.6-103.3% recovery rates and 0-4.61% relative errors.