A sequential injection (SI) separation system has been developed for separation and analysis of Am, Pu, and Np isotopes using on-line inductively coupled plasma mass spectrometry (ICP MS) detection. On-line actinide separations were carried out using an actinide-specific extraction chromatographic material (TRU-resin, Eichrom Industries, Inc. USA). Separations are demonstrated and characterized that are compatible with on-line ICP MS detection and address isobaric ( 241 Am/ 241 Pu, 244 Pu/ 244 Cm, 238 U/ 238 Pu), molecular ( 238 UH/ 239 Pu), and spectral ( 238 U/ 237 Np) interferences encountered in the analysis of Am, Pu and Np isotopic composition using ICP MS. The effects of reductive sample treatment on the extent of U matrix removal were investigated. Uranium separation factors were limited by the formation of U(IV) species, which exhibited separation behaviors similar to that of Pu(IV) and Np(IV). Nevertheless, separation factors as high as 3.0 3 10 5 can be achieved at the 0.1 mg ml 21 U concentration levels using Fe(II) sulfamate as a reductant. The sequential injection (SI) ICP MS technique has been applied towards analysis of Am, Pu, and Np isotopes in a dissolved vitrified nuclear waste sample. Results obtained using the automated separation technique with ICP MS detection were in satisfactory agreement with results obtained using standard analytical methodologies.