Abstract: To optimize the synthesis and purification process of everolimus, and to investigate the quality of everolimus, synthetic schemes were designed to prepare 2 related substances, resulting from the process of everolimus. These two related substances were 28, 40-O, O′-bis(2-hydroxyethyl)rapamycin and 28-O-(2-hydroxyethyl)rapamycin. Their structures were confirmed by ¹H NMR and MS, and their presence in everolimus was confirmed by HPLC.