Colored microspheres have become popular compared with radioactive microspheres because they do not use radioactivity. However, they suffer from a much greater variability in their determination. We have developed a new method for assaying the dye using high-performance liquid chromatography (HPLC) with internal standard. This technique permits accurate determination of 400 spheres in rat blood, heart, kidney, liver, and brain with a relative error [coefficient of variation (CV)] <10%. To date, only three colors (white, yellow, and red) may be used because, of the five colors tested, one (violet) served as internal standard and another (blue) exhibited marked degradation during extraction. Compared with the classical spectrophotometric technique, HPLC allows a three to five times improvement in reproducibility with a relative error significantly lower (P < 0.01) than with direct spectrophotometry. Although this new technique appears to be more time consuming than the classical method, its use seems to be preferable because of the improvement in measurement sensitivity.