Double-focusing sector field inductively coupled plasma mass spectrometry (ICP-MS) was used for the determination of ²⁶Mg/²⁴Mg isotope ratios in order to quantify intestinal Mg²⁺ transport in the fish tilapia (Oreochromis mossambicus). As the sample medium contained substantial amounts of C, Na, P, S, Cl and K, the occurrence of molecular (e.g., C₂⁺, C₂H⁺, CN⁺ and NaH⁺) and doubly charged (⁴⁸Ca²⁺) ions and tailing of the ²³Na⁺ ion signal resulted in spectral overlap at low mass resolution. Application of a higher mass resolution (R = 3000), however, permitted accurate determination of Mg isotope ratios. The usefulness of cool plasma conditions was also evaluated, but this approach was insufficient to resolve all interferences completely. After careful optimisation of the measurement parameters, it was possible to measure the ²⁶Mg/²⁴Mg isotope ratio with a relative standard deviation (RSD) of 0.2–0.5% (n = 10 × 30 s) at R = 3000. From the isotope ratio results obtained, net intestinal Mg²⁺ fluxes were calculated, providing evidence for secretory Mg²⁺ transport. Conclusively, the measurement of stable isotope ratios with sector field ICP-MS presents a sophisticated tool for the study of Mg²⁺ transport processes in biological systems that may provide an effective alternative for the use of radiotracers.