The extraction and isolation of polychlorinated biphenyls (PCBs) from sample matrices such as freeze-dried milk is a lengthy and expensive process that conventionally requires the isolation of microgram amounts of PCBs from large volumes of flammable organic solvents used in the extraction and chromatographic stages. Supercritical-fluid extraction (SFE) and chromatography (SFC) using carbon dioxide has been investigated as a replacement procedure using a Simplex optimisation approach to optimise the working conditions. The SFE conditions required to extract PCBs and milk fat from freeze-dried milk were investigated, and it was found that although extraction was rapid (10 min), the conditions required were identical so that there was no separation of PCBs and fat. However, SFC conditions have been developed that permit the resolution of PCBs from milk fat in 15 min. Thus, a combination of SFE and SFC have the potential to replace the existing methods of extraction and isolation with procedures that are quicker, cheaper, and avoid the use of flammable organic solvents.