<p>Troponin activators, including Reldesemtiv and Tirasemtiv, represent a class of drugs that enhance the contractility of cardiac muscle and delay the onset of muscle fatigue. Consequently, they were classified as prohibited substances by the World Anti-Doping Agency (WADA) in 2024. This study aimed to develop an ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS)–based method to quantify Reldesemtiv and Tirasemtiv levels in rat urine, blood, and feces over a 72-h period following transgastric administration. Additionally, Compound Discoverer 3.3 was utilized to investigate the metabolites and metabolic pathways of these two drugs in vivo. Twelve metabolites of Reldesemtiv and seven metabolites of Tirasemtiv were identified. Notably, ten of the twelve Reldesemtiv metabolites had not been previously reported in the literature. Using UHPLC-MS, the concentration of each metabolite in urine and blood was measured at various time points, enabling the generation of a metabolic profile. Six metabolites of Reldesemtiv are detectable 72&#xa0;h after administration, while Tirasemtiv and its metabolite T-M1a were also detectable at the same time point. This analytical method can characterize the metabolic profiles of the drugs and their metabolites, as well as identify unknown and long-lived metabolites in drug testing. These capabilities are critical for the quantitative analysis of troponin activators within complex biological matrices, supporting their study in physiological and doping-control contexts.</p>