Context: Iron is an essential element for most life on Earth, including human beings and its accurate determination is extremely important. A simple and sensitive spectrophotometric method has been used for the determination of iron concentration in blood serum and compare with atomic absorption spectrophotometric method. Objective: To find the appropriate, simple and accurate methods for iron determination in serum samples Design: A comparative study between two analytical methods. The methods are based on the reaction of iron with ferene reagent at acidic condition. The method of determining iron in serum by flame atomic absorption spectrophotometry with slurry nebulization into an air-acetylene to various flame has been used. Main Outcome Measure: Serum iron concentrations in different serum samples. Results: The results showed that distribution of iron content between serum samples are different, it was between 41 to 343.5 mcg/dL. Values of iron content measured in the serum samples with spectrophotometer method are in good agreement with the results obtained with atomic absorption method. Conclusions: The spectrophotometric method with ferene reagent can be successfully and accurately applied to the determination of iron in serum and it can be used instead atomic absorption.