Bisphosphonates are approved by the US FDA in horses older than 4 years of age and are of concern to the equine racing community due to their ability to provide analgesia while inhibiting the breakdown of bone by osteoclasts. They are highly polar and have poor ionizability thus making them difficult to detect with mass spectrometry and requiring extensive sample preparation. Therefore, an alternative approach using untargeted metabolomics and lipidomics was performed to identify potential biomarkers associated with the administration of bisphosphonates within equine athletes. An eight-horse case-control OsPhos® administration study was conducted over a 12-week period. Six metabolomics and lipidomics workflows were implemented to analyze the plasma samples, in a randomized manner, for optimal metabolome coverage. Compound Discoverer was used to detect and identify features using metabolomics and lipidomics algorithms and library searching, such as mzCloud, ChemSpider, BioCyc, the Human Metabolome Database and the Kyoto Encyclopedia of Genes and Genomes. Metaboanalyst 6.0 was used to visualize the data and identify statistically significant biomarkers. Compound Discoverer was able to reduce the many features within the six workflows to those which were actual peaks. Data transformation reduced bias within the data, allowing for the discovery of significant biomarkers. These biomarkers that experience a smaller fold change may go undetected without transformation due to the impact from biomarkers skewing the data. Four of the six workflows achieved separation between control and treated horses with good accuracy and predictability through orthogonal partial least squares-discriminant analysis models. Potential biomarkers were identified using a fold-change cut off greater than 2.0, a p-value of less than 0.05 and a Variable Importance in Projection score greater than 1. A resulting 124 potential biomarkers were established from five of the six workflows for further investigation using a second independent set of horses in the OsPhos® administration study.