Enzymatic assays need robust, rapid colorimetric methods that can follow ongoing reactions. For this, we developed a highly accurate, multi-wavelength detection method that could be used for several systems. Here, it was applied to the detection of <i>para</i>-nitrophenol (<i>p</i>NP) in basic and acidic solutions. First, we confirmed by factor analysis that <i>p</i>NP has two forms, with unique spectral characteristics in the 240 to 600 nm range: Phenol in acidic conditions absorbs in the lower range, whereas phenolate in basic conditions absorbs in the higher range. Thereafter, the method was used for the determination of species concentration. For this, the intensity measurements were made at only two wavelengths with a microtiter plate reader. This yielded total dye concentration, species relative abundance, and solution pH value. The method was applied to an enzymatic assay. For this, a chromogenic substrate that generates <i>p</i>NP after hydrolysis catalyzed by a lipase from the fungus <i>Yarrowia lipolytica</i> was used. Over the pH range of 3-11, accurate amounts of acidic and basic <i>p</i>NP were determined at 340 and 405 nm, respectively. This method surpasses the commonly used single-wavelength assay at 405 nm, which does not detect <i>p</i>NP acidic species, leading to activity underestimations. Moreover, alleviation of this pH-related problem by neutralization is not necessary. On the whole, the method developed is readily applicable to rapid high-throughput of enzymatic activity measurements over a wide pH range.

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