Fluopyram is a fungicide and nematicide that belongs to the chemical group of benzamides, which act as succinate dehydrogenase inhibitors (SDHIs) on the mitochondrial respiratory chain of fungi. Despite being well known in several countries, there are few studies involving the optimization and validation of extraction methods for determining fluopyram in water samples. Therefore, this study aimed to optimize and validate liquid-liquid extraction with low-temperature purification (LLE-LTP) to determine fluopyram fungicide in water samples using high-performance liquid chromatography with diode array detection (HPLC-DAD). A two-level full factorial design was employed to optimize LLE-LTP which enabled achieving a recovery rate close to 100% and relative standard deviations (RSD) < 10.0%. The validation showed that the extraction method may be considered selective, precise, accurate, and linear in the range of 6.0 to 200 μg L-1. The LOD and LOQ were 4.0 and 6.0 μg L-1, respectively, proving the efficiency of this method for trace level determination of this fungicide in water samples. LLE-LTP coupled to HLPC-DAD analysis showed a matrix effect of less than 8% and it was applied in monitoring 20 environmental water samples, but no fluopyram residue was detected in the samples.