Quantification of fatty acids (FA) in reproductive tissues including the placenta is important to understand the influence of FA on reproductive function. Two methods of FA determination (direct and indirect) are commonly used, but their comparative efficacy to quantify FA in bovine tissue is not known. Therefore, we compared the efficacy of the 2 methods of FA determination in bovine placental tissue. Placenta (fetal cotyledon) samples from 13 dairy cows were collected within 5 h of calving, before placental release, snap frozen and stored. Tissues (0.05 g) were assigned in duplicate to either direct methylation (samples directly subjected to methylation process with no extraction step [direct method]) or indirect methylation (samples first subjected to FA extraction and then methylated [indirect method]). The indirect method was approximately 6 h long that involved an 18-step procedure, whereas the direct method took 3 h and had only 10 steps. Briefly, frozen tissue samples were pulverized under liquid nitrogen and methylated either directly, without extraction, or indirectly, after extraction. The FA methyl esters were then injected for gas chromatographic analysis. Fatty acid data were analyzed using the Mixed procedure of SAS. Forty-five different FA were identified from the placental tissue, of which 32 FA were significantly higher in the indirect method. Moreover, the unsaturated-long chain FA of our interest, i.e., oleic, linoleic, α-linolenic, eicosapentaenoic and docosahexaenoic acid were significantly higher (P < 0.0001) in the indirect method than in the direct method (0.42, 0.08, 0.008, 0.02 and 0.05 vs. 0.30, 0.06, 0.005, 0.01 and 0.04 mg per 50 mg sample, respectively). In addition, the total amount of FA, saturated FA, polyunsaturated FA, omega-3 and −6 FA were also higher (P < 0.0001) with the indirect method than the direct method. Even though, the direct method is shorter and faster, it reduced the amount of FA extracted. We conclude that the indirect method has higher efficacy and should be preferred over the direct method for extraction of FA from placental tissue.