A new approach has been proposed for determining the particle size of the sulfide phase of hydrotreating catalysts. The method includes sulfiding the catalyst with hydrogen sulfide containing the S-33 nuclide; carrying out the thiophene hydrodesulfurization reaction; and, at the final stage, determining the isotopic composition of sulfur on the catalyst using a combination of temperature-programmed oxidation and mass spectrometry techniques (TPO-MS). It has been shown that during hydrodesulfurization, part of the most labile sulfur S-33 on the faces of hexagonal prisms of MoS2 is replaced by S-32 from thiophene, and the degree of this substitution can be determined by the TPO-MS method. It has been found that with a decrease in the particle size of the sulfide phase, which is confirmed by transmission electron microscopy (TEM), the proportion of labile sulfur capable of substitution during hydrodesulfurization increases.