Background: Glucose is an essential element in the supply of body's energy. In diseases such as diabetes, glucose increase is associated with disturbance in metabolism. Cortisol is an important hormone in the regulation of glucose metabolism, and human serum albumin (HSA) is one of the most important glucose and cortisol transmitters in blood. Interaction between albumin and these ligands could affect HSA secondary structure and its stability. Objectives: The aim of this study was to investigate HSA secondary structure in the presence of different concentrations of glucose and cortisol. Methods: This was an in vitro (analytical/descriptive) study in which, completely randomized design was used to study the interaction between human serum albumin