Tobramycin is an aminoglycoside antibiotic that shows antimicrobial activity against a wide spectrum of aerobic bacteria. This paper describes the electrospray ionization mass spectrometric analysis of metallo (Cu(II) and Fe(III))–tobramycin complexes. The Cu(II) (1.5 × 10−4 M) and Fe(III) (1.5 × 10−4 M) ions co-ordinate with tobramycin (1.5 × 10−4 M) at 1:1 molar ratio for 30 min at room temperature. ESI mass spectrum of tobramycin shows ions at m/z 468.1, 934.8, and 234.8 corresponding to mono-protonated ([M+H]+), dimer ([2M+H]+) and di-protonated ([M+2H]2+), respectively. The Cu(II) and Fe(III) complexes show preferentially different molar ratio as 1:1, 1:2 and 1:3 such as [63Cu+(M−2H)+H]+, [65Cu + (M−2H) + H]+, [63Cu + (M−2H) + 2H2O + H]+, [65Cu + (M−2H) + 2H2O + H]+, [Fe + (M−3H) + 2H2O + H]+, [Fe + (2M−3H) + 2H2O + H]+, [Fe + (M−3H) + H2O + H]+ and [Fe + (M−3H) + H]+, respectively. Therefore, ESI-MS provides a simple, accurate and reproducible method for the identification of Cu(II)– and Fe(III)–tobramycin complexes ions than the other conventional methods (the method of continuous variations, the mole-ratio method, the slope-ratio method, computer-based curve-flitting methods).