Determining the use of off-label bisphosphonates (BPs) in young horses (< 4 years old) is of interest due to the threat to racing integrity and welfare associated with this class of drugs that have the potential to manipulate bone structure. The analysis of BPs in biological matrices is challenging by liquid chromatography coupled to mass spectrometry (LC-MS/MS) and alternative sample preparation approaches are needed to detect their misuse in racehorses. There are currently two classes of BPs: non-nitrogen and nitrogen-containing with each having a distinct mode of action. Currently two non-nitrogenous based BPs are available for use in horses, tiludronic acid (or tiludronate) and chlodronate for the treatment of navicular syndrome (Mitchell et al. BMC Veterinary Research 2019;15(1):211) in horses over the age of 4. There is, however, evidence that BPs can persist in the bone for several years after administration since they can form hydroxyapatite crystals prior to being fully absorbed by active osteoclasts where full inhibitory action is performed (Riggs et al. Equine veterinary journal 2021;53(6):1287–1295). Therefore, with this prolonged detection time for the BPs, it is necessary to investigate whether the use of metabolomic biomarkers can provide a complementary method for the detection of their misuse. The aim of this research was to develop a biomarker approach for complementary direct detection of bisphosphonate drugs in equine plasma. BPs are hydrophilic therefore the targeted detection of tiludronic acid in equine plasma required an orthogonal solid phase extraction (SPE) method using both a polymeric reversed-phase sorbent and a weak anion exchange cartridge prior to derivatisation using trimethyl orthoacetate and reverse LC-MS/MS. Endogenous lipids and corticosteroids in equine plasma were detected after protein precipitation and SPE with a polymeric reverse phase cartridge prior to liquid chromatography high-resolution mass spectrometry. Data was analysed using the Shimadzu Insight Explore software to determine up- or down-regulation of biomarkers. Results were then profiled using a Parametric Empirical Bayes algorithm to propose individual reference limits (IRLs). Tiludronic acid was detected up to the final collection point of 28 days post-administration. Following a targeted approach, up- and down-regulation of the eicosanoid and corticosteroid ratios were observed with the corticosteroid ratio exceeding both the proposed population reference limit (PRL) and IRL. Using a semi-targeted approach, several additional lipids were identified to have activity during the administration and will require further confirmatory analysis. Monitoring the impact on endogenous biomarkers following an administration of Tiludronic Acid in racehorses is limited therefore little is known if BPs have a direct or indirect effect on the inflammatory pathways. However, research in humans has linked BPs with possible effects of analgesia in addition to bone remodelling that BPs are primarily used for. Therefore, it is necessary to further extend detection capabilities to ensure endogenous eicosanoids and corticosteroids are monitored. This, however, should not negate the need for the detection of the drug itself as tiludronic acid has the potential to be detected as far as 28 days post-administration. Therefore, a complementary biomarker approach would be preferable for the detection of non-nitrogen-based BPs, which can be further applied for the nitrogen-based BPs. A complementary biomarker approach to direct detection of administered drugs can improve the understanding of effect and support non-targeted detection strategies for analogues of specific drug classes.