A newly developed large-volume injection (LVI) technique that employs a unique stomach-shaped inlet liner (SSIL) inside of a programmable temperature vaporizer was used for the determination of trace amounts of dioxins in human milk and plasma. The initial temperature and the initial dwelling time of the inlet and the kind of solvent used were found to be critical in determining the analytical sensitivity of dioxins due to the loss of these relatively volatile compounds during solvent vaporization. Human milk and plasma were purified and fractionated by pre-packed multi-layered silica-gel chromatography and activated carbon silica-gel column chromatography. A 20-µL aliquot of the fraction collected from the chromatography with toluene was directly applied to the LVI system in high-resolution gas chromatography/high-resolution mass spectrometry. Excellent correlation (r > 0.97) between the values obtained by the LVI method using the SSIL device and those by the conventional regular-volume splitless injection method was obtained for PCDDs, PCDFs and non-ortho PCBs in human milk and plasma samples.