Biochemical markers are used to investigate food web structure of many ecosystems and to describe the trophic ecology of an increasing number of marine taxa. Fatty acids (FA) profiles are a valuable tool to study trophic interactions between marine organisms. However, functional limitations and capabilities of these biomarkers must be assessed within the context of species-specific collection methods. We analysed FA in muscle tissue biopsies through non-lethal sampling of white shark (Carcharodon carcharias) and investigated the minimum sample size needed to provide reliable measurements of FA profiles and the effects of sample degradation over time. Muscle samples as small as 12 mg DW provided reliable and consistent FA profiles, while connective tissue less than 40 mg DW yielded inconsistent profiles. Both amounts validate the suitability of FA analysis for use with punch biopsies, and suggest the methods could be used with scalpel biopsies of smaller elasmobranchs. The integrity of FA profiles in muscle and connective tissue samples were retained for the first three days, with differences in FA profiles appearing over the following two days, including relative decreases in key FAs, making these degraded samples unreliable for accurate determination of dietary sources. Together, these investigations determined the functional limitations of FA analysis within the context of tissue collection methods available for a protected species from which tissue samples are logistically difficult to obtain. This study provided key information to ensure reliable FA profiles can be obtained to assess the feeding ecology of high-order predators, including the white shark.