This review article summarizes the results of the use of UV absorption spectroscopy in the studies of the photoisomerization of provitamin D in vitro. It was the spectral observations of the progress of the photoreaction that revealed anomalous kinetics upon laser irradiation at λ = 308 nm that contradicted the standard model. A well-grounded approach to explaining the wavelength dependence of the kinetics and maximum accumulation of previtamin D is presented based on a comparison of the well-known UV absorption spectra of provitamin D and its photoisomers in a solution. An original approach to spectrophotometric concentration analysis of a multicomponent mixture of provitamin D photoisomers, considering its photodegradation, is mentioned, and the significant consequences of the discovered effect for the industrial synthesis of vitamin D and biological UV dosimetry are discussed. In conclusion, the influence of the microenvironment on the conformation distribution of the previtamin D molecule and the kinetics of its photoconversions is considered.