Purpose To investigate the precipitation process of a mixture of vancomycin, amikacin, and dexamethasone by equilibrium dialysis and its subsequent effect on the levels of available-free antibiotics and steroid. Methods Concentrations of amikacin, vancomycin, and dexamethasone in an equilibrium dialysis chamber were measured during the equilibrium process by highperformance liquid chromatography and fluorescence polarisation immunoassay. Vitreous were used as the medium of dialysis, with the three medications prepared in normal saline (NS) and balanced salt solution plus (BSS Plus) separately. Results Amikacin showed no measurable loss in NS or BSS Plus, either alone or when mixed with vancomycin or dexamethasone. Vancomycin showed minimal loss in BSS Plus, either alone or when mixed with amikacin or dexamethasone. Dexamethasone showed a median loss of 16 and 15% when incubated alone in NS and BSS Plus, respectively, at 48 h. When mixed with vancomycin or amikacin in BSS Plus, it showed a median loss of 13 and 12%, respectively, at 48 h. There was no statistically significant difference in the loss of dexamethasone under various conditions. In equilibrium dialysis in vitreous, amikacin, vancomycin, and dexamethasone reached equilibrium within 24 h and with no loss up to 192 h. There was no difference observed when the medications were prepared in NS or BSS Plus.Conclusions Both amikacin and vancomycin did not show precipitation or decrease in concentration in NS or BSS Plus. Dexamethasone showed relatively small percentage loss. As a result, treatment of endophthalmitis with vancomycin and amikacin combination is preferred.