A novel screening experiment, to find radioactive probes for non-invasive measurements of physiological functions in experimental animals, was tested using the in vivo multitracer analysis technique. The details of the efficiency of the detector settings used in the in vivo multitracer analysis technique were examined by both computer simulations and practical measurements. Multiple radioactive isotopes, i.e. multitracer, were prepared by irradiating a silver foil target with a heavy ion beam at the RIKEN ring cyclotron. After chemical separation of the silver target, the multitracer was finally dissolved in isotonic citrate buffer. The multitracer solution was intravenously injected into rats. Using a γ-ray detector equipped with a well-defined slit, the collimated γ-rays from the upper abdomen of living rats were measured. After correction of detection efficiencies, it was possible to compare the distribution of radioactive elements between two groups of rats different in body weight. The in vivo measurement showed that the tissue substantial volume of the selenium-deficient (SeD) rat liver increased compared to normal rats. The possibility of a functional estimation of tissue/blood volume for living rats was proposed based on the characteristic in vivo distribution of ⁷⁴As, ⁸³Rb and ¹⁰³Ru.