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Feline Infectious Peritonitis (FIP) has been transformed from a fatal disease to a treatable condition following the introduction of GS-441524, a nucleoside analogue targeting feline coronavirus replication. However, the widespread use of unregulated compounded formulations and the absence of validated analytical tools for therapeutic drug monitoring (TDM) represent critical gaps in clinical FIP management. This study describes the development and full ICH M10-compliant validation of a high-throughput LC-MS/MS method for the quantification of GS-441524 in feline serum, incorporating an automated protein precipitation protocol and a PBS-BSA surrogate matrix in accordance with 3Rs principles. The method met all acceptance criteria across validated parameters, including linearity (0.1–50 µg/mL), accuracy (bias within ±12.5%), precision (CV ≤10.9%), selectivity, extraction recovery (87.5–107.9%), and stability under clinically relevant storage conditions. Matrix equivalence between PBS-BSA and authentic feline serum was confirmed, enabling routine calibration without animal-derived materials. The validated method was applied to clinical TDM in cats undergoing GS-441524 treatment for FIP, providing preliminary evidence of inter-individual pharmacokinetic variability. The compounded formulations administered to the TDM cohort were independently verified by LC-MS/MS, confirming drug content within ±15% of labelled claims and excluding pharmaceutical quality as a confounding factor in the interpretation of serum drug concentrations.