A novel spectrophotometric method for determination of chloramphenicol (CAP) based on the diazotization reaction at room temperature has been developed. The CAP was reduced using zinc powder and diazotization reaction was carried out at room temperature in the presence of NaNO2, bismuth nitrate pentahydrate as catalyst. 2-napthol was used as coupling agent to forma red-violet solution and the absorbance was measured by UV-Vis spectrophotometer at 554 nm. The analytical parameters including optimization of reducing agent, catalyst, coupling agent and time response for formation of azo dye compounds had been investigated.