Spectrophotometry, a branch of spectroscopy, focuses on measuring optical properties of matter as a function of wavelength and on the interaction of matter with electromagnetic radiation. Different types of spectrophotometry include UV–VIS, IR, fluorescence, atomic absorption and atomic emission spectrophotometry, each suitable for specific applications. Because of their ability to precisely identify and quantify compounds, spectrophotometers are widely used in environmental analysis and in numerous fields of science and human activity. In addition to an overview of spectrophotometric principles and methods, this research focuses on two experimental goals: evaluation of microbiological degradation of azo dye Ponceau S and evaluation of quality and authenticity of olive oil. For the Ponceau S decolorization analysis, samples were prepared in three different media with Pseudomonas aeruginosa suspension and a 100 mg/L dye solution, and absorbance in samples was measured at different times using a VIS spectrophotometer. Microbiological degradation of azo dye occurred, and spectrophotometric measurements determined the degree of discoloration. For the analysis of olive oil, absorbance of six homogenized samples of olive oil in isooctane was measured at certain wavelengths using the prescribed method and a UV-VIS spectrophotometer, in order to detect oxidation products that indicate the quality of oil. Results exclude the possibility of adulteration of oil and indicate inadequate storage conditions. Methods described and results obtained from this research show the effectiveness and versatility of spectrophotometry in the analysis of complex samples.