Albumin has a fundamental role in human body. Its main tasks in blood are to regulate osmotic blood pressure, maintaining the pH, and transporting metabolites and drugs throughout the vascular system. Pharmacological studies of the interaction of drugs on HSA are important due to structural and functional changes of this vital protein; thus, here in this research the effect of valproate as a common drug for epilepsy disorders is evaluated in the presence of hexadecyl pyridinium bromide (HPB) as a positive surfactant in normal and fever condition. Electrochemical method was used to investigate the binding number of HPB molecules to HSA in the absence and presence of valproate by evaluating the concentration of free HPB in 37oC and 42oC temperatures. HSA affinity for valproate binding studied via ligand binding process for normal and fever temperatures. The findings indicate that, there is a significant difference in valproate binding to albumin at physiological and pathological temperatures. The consequences are the same in the presence of HPB; in other words, HSA binding tendency to HPB in the presence of valproate was totally altered because of HSA major conformational changes in fever condition. In conclusion, corrected dosage of valproate is needed for fever condition relative to normal temperature and the patients under prescription of different medications in fever condition should have different orders due to the interferences of drugs.