A highly selective and sensitive derivative spectrophotometric method has been developed for the determination of Fe(II) and Ni(II) in different mineral vitamins. The method is based on the formation of binary complexes of Fe(II) and Ni(II) with 2,4-diamino-5-(2-hydroxy-5-nitrophenylazo) benzenesulfonic acid sodium salt (Mordant Brown 33) at pH 5.6 with Tween 20. At lmax 512 and 493 nm, the molar absorbtivity was 2.09 ´ 104 and 0.58 ´ 104 L/mol cm for Fe(II) and Ni(II), respectively. Fe(II) can be determined in the range 0.55–2.79 mg/mL in the presence of 2.92 mg/mL Ni, and Ni(II) can be determined in the range 1.17–3.5 mg/mL in the presence of 2.79 mg/mL Fe(II) in the presence of Tween 20 (4%). The detection limits were 9.0 and 15.0 ng/mL for Fe(II) and Ni(II), respectively. The effect of foreign ions was elucidated. The RSD values were, in all instances, less than 1.3%. The proposed method was successfully applied for the simultaneous determination of Fe(II) and Ni(II) in different mineral vitamins.