A method for investigating the distribution of therapeutic and diagnostic substances inside nanoparticles was developed for spherical amorphous nanoparticles (SANPs). SANPs were generated from the lupan triterpenoids of birch bark (LTs). This method was based on the sequence of short exposures of SANPs in a special solvent. The thin surface layers of the main material and the material of substances incorporated into the SANPs were dissolved. The distribution of three different substances in the SANPs were analyzed by determining the optical density of the solutions containing material extracted from SANPs. It was shown that the distribution of therapeutic and diagnostic substances in SANPs depended on their solubility in the solution saturated with the main SANPs material and their mass fraction. © 2010 Pleiades Publishing, Ltd.