An increasing number of studies use blood obtained noninvasively to monitor organohalogen contaminants; however, blood can be difficult to analyze because of its aqueous nature and high protein content. We compared five methods for extracting polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides, and lipids from serum using National Institute of Standards and Technology Standard Reference Material 1589a PCBs, Pesticides, PBDEs, and Dioxins/Furans in Human Serum. Liquid:liquid (LLE), cavity-dispersed microwave-assisted (MAE), focused microwave-assisted (FME), solid-phase (SPE), and pressurized fluid (PFE) extraction techniques were compared. FME extraction yielded the optimal recovery of internal standards (IS). All methods resulted in similar contaminant concentrations that also agreed with the certified values for SRM 1589a, except for only a few compounds measured by methods other than FME. Based on these findings, the FME method was determined to be the best overall extraction method. One procedural factor was found to affect contaminant concentrations; use of IS carrier solvents that were immiscible with serum (or when the serum was not directly physically mixed with IS) resulted in a 30% underestimation of organohalogen concentrations. This study offers valid, novel extraction alternatives beyond traditional methods (e.g., LLE) for blood contaminant measurements.