The analytical method used for the determination of polychlorinated biphenyls (PCB), polychlorinated dibenzo-p-dioxins (PCDD), and polychlorinated dibenzofurans (PCDF) in human milk comprised extraction of lipids, the use of 51 13C12-labelled PCB and PCDD/PCDF as internal standards, several chromatographic purification steps, and high resolution gas chromatographic/high resolution mass spectrometric measurement. As an accredited laboratory since 1998, a comprehensive quality control programme has been applied to assure the long-time reliability of results of human milk samples received for WHO/UNEP-coordinated exposure studies between 2000 and 2019. This included procedural blanks, the use of fortified vegetable oil and numerous quality control samples as an in-house reference material, duplicate analyses, and successful participation in 32 proficiency tests (PTs) covering 81 samples of food of animal origin or human milk. Trueness was estimated from the PT samples in the relevant range for human milk above 1 pg WHO-TEQ/g lipid: The deviation was less than 10% from the assigned values for WHO-PCDD/PCDF-PCB-TEQ and WHO-PCDD/PCDF-TEQ and less than about 15% for WHO-PCB-TEQ for about 90% of the results. For the sum of six non-dioxin-like PCB (relevant occurrence range, 1–1000 ng/g lipid), approximately 90% of the results differed by less than 15% from the assigned values. A long-term precision of <15% (coefficient of variation of within-laboratory reproducibility) was achieved, based on quality control samples analysed between 2000 and 2019.The analytical methodology used fulfilled the requirements of the general criteria for the operation of testing laboratories as laid down in EN ISO/IEC 17025, the analytical criteria for PCDD/PCDF and PCB in feed and food specified in EU legislation, and the criterion for monitoring information for Parties to the Stockholm Convention.