Background: The bioaccumulation potential of chemical substances is commonly determined in flow-through fish tests according to the Organization for Economic Cooperation and Development (OECD) technical guideline (TG) 305, aiming at the determination of bioconcentration factors (BCF). Investigations on aquatic organisms have shown a significant positive correlation between the accumulation of a chemical and the lipid content of organisms. Residue levels can thus differ between individuals, species, and size groups depending on their lipid content. BCF values can be normalized by lipid content; however, this requires the use of appropriate extraction techniques to ensure the complete extraction of total lipids from fish/tissue samples. The OECD TG 305 is currently under revision. A literature review was carried out to select preferred methods which shall be included in the updated OECD TG 305 along with a sampling schedule for lipid measurement in fish. Results: An overview and appraisal of a broad range of lipid extraction methods is presented. Extraction procedures applied in bioaccumulation studies described in the literature and procedures currently used for such studies by governmental, academic, and industrial labs are surveyed. Possible impacts of unsuitable extraction techniques on lipid normalized BCF results were assessed. Default values for BCF normalization commonly used in bioaccumulation studies are described. The effect of differences in residue distribution over different organs and/or lipids on the normalization of BCF values is discussed. Conclusions: A broad range of procedures for lipid extraction are available. However, only the use of suitable extraction techniques guarantees the complete extraction of total lipids from fish samples in bioaccumulation studies which is required to ensure the correct lipid normalization of BCF values. Preferred methods for inclusion in the updated OECD 305 test guideline as well as a standard schedule for lipid measurements are suggested.