Drug-eluting stents have attracted significant attention in the medical community and pharmaceutical industry due to their proven success in significantly reducing restenosis. Abbott Laboratories is developing a drug-eluting stent coated with zotarolimus and swine was recently used as an animal model for the pre-clinical study of stent implantation. In this article, we present a detailed experimental design and results for the validation and sample analysis of zotarolimus drug concentration in stented swine artery samples. Introduction of tissue quality control (QC) samples allows evaluation of the entire analytical process as well as the stability of the drug in both original tissue and homogenized tissue samples. In addition, a novel approach using 100% swine blood as the homogenization solution was developed for the consistency of the liquid–liquid extraction recovery and stability of the zotarolimus in tissue homogenates. Standards were prepared by spiking zotarolimus working solution in swine blood and tissue QC samples were used along with the artery samples during the sample analysis. The linear dynamic range of blood standard samples is from 0.61 to 333.20 ng/mL to accommodate the predicted artery homogenate concentrations. Overall tissue QC %CV during the method validation was from 4.4% to 8.6%. The overall %bias of tissue QC samples during the method validation was from −7.3% to 16.6%. The method was successfully applied for the analysis of swine artery samples. A similar approach for method validation and sample analysis has been successfully applied for the analysis of swine myocardium, kidney and liver tissue samples. Copyright © 2006 John Wiley & Sons, Ltd.