A method for the determination of the total lipid content in fish meat was established using a 2-thiobarbituric acid (TBA) reaction, which had previously been used for the determination of lipid peroxides in animal tissues. In this method, an unspecific peroxidation of fish oils was created by omitting the addition of antioxidant to the reaction mixture during the TBA reaction, because fish meat is more sensitive to the TBA reaction due to its higher concentration of polyunsaturated fatty acids, especially docosahexaenoic acid and eicosapentaenoic acid, which can easily react artificially with TBA in the absence of an antioxidant in the assay system. As for a practical application of this method, we tried to optimize the assay procedures in the sampling, reaction, and detection steps of this method, and finally proposed a new standard procedure recommended for determining the total lipid content of fish using a TBA reaction. In order to confirm the accuracy of the new procedure, comparative evaluations for the lipid contents of commercially available fish, i.e., chub mackerel (Scomber japonicus) and saury (Cololabis saira) were made between the conventional procedure and the recommended TBA method. The lipid contents obtained by the two methods coincided well with high correlation. This method is relevant for total lipid content analysis of fish meat under restricted laboratory conditions.