The concentration of oestradiol in follicular fluid or its ratio to progesterone is often used to identify atresia in follicles. However, the value of this test is limited because in healthy small antral follicles and very large mature follicles approaching ovulation, the levels of oestradiol are either low or declining. Therefore, there is a need for better biomarkers of atresia in follicular fluid. We examined the composition of follicular fluid from morphologically healthy and atretic small (3-5 mm), medium (6-9 mm) and large (> 10 mm) follicles in the cow (n = 6 per group). Samples were extracted with methanol/chloroform to remove proteins > 1 kDa, and the polar and non polar fractions were subjected to HILIC (Hydrophilic Interaction) and Reverse Phase (RP) Liquid Chromatography / Mass Spectrometry (LC/MS) analysis, respectively. Total ion chromatograms were normalised in mzMine (v2.3) to identify possible isotopes, adducts and ion complexes of the same molecule. Extracted ion chromatograms for individual features of a particular mass were generated and used to consolidate the data into fewer peak groups with a higher degree of confidence. Principal Component Analysis (PCA) was conducted. Median fold change in peak intensity and Student’s t-test were used to analyse the data further. For the HILIC data, PCA was largely able to separate the three healthy groups of the different sizes. More molecules were determined to be significantly different between healthy and atretic groups at each size for both the HILIC (n=146) and the RP (n=106) analysis, particularly for the medium size follicles. Molecules identified as down regulated on atresia include several species of lysophosphatidylcholine (Lyso-PC). Molecules up regulated in medium atretic follicles include omega fatty acids and the steroid metabolites. Results indicate that follicular atresia is accompanied by distinct changes in the metabolomic profile useful for identifying better biomarkers of atresia.