The aim of this study was to develop a chromatographic method for 4-NP determination in anaerobic batch reactors using low amount of organic solvents and also evaluate 4-NP degradation in the presence of different co-substrates. Chromatographic parameters was improved for stationary phase (C-8 column), mobile phase (acetonitrile/water solution 90:10, v:v), column temperature (40 ºC), eluent flow (0.8 mL min-1), injection volume (100 µL) and wavelengths in fluorescence detector (FLD) (λex=225 nm and λem=305 nm). Solid phase extraction (SPE) was performed for sample purification with smaller amount of solvents (methanol - 2 mL and ethanol - 1 mL) than previously reported. Reduction in reagent consumption was about 5.5 times for HPLC and about 19 times for SPE, according to previously reported. Method was validated according to the linearity (0.9951), selectivity and limits of detection and quantification were 15.7 µg L-1 and 55.6 µg L-1, respectively. The 4-NP removal in anaerobic batch reactors was higher than 50% (4-NP initial concentration of 398 µg L-1). Fumarate was a better co-substrate to favor methanogenesis in the presence of 4-NP, which provided higher organic matter removal, higher methane production speed and shorter start-up time for methane production.