The ability to directly compare gastric ulcerated and healthy tissue would aid in the understanding of the physiological differences between these tissue types. Presently, these comparisons can only be drawn by the use of separate animal groups, which results in increased study variability. The focus of this research was to develop a four-probe microdialysis sampling approach to directly compare ulcerated and healthy tissue in the same animal. After controlled chemical ulcer induction, probes were implanted in the ulcerated and healthy stomach submucosa, stomach lumen and in the blood. To assess the significance of this multiple probe approach, drug concentrations in each probe location were monitored after selected compounds were dosed to the ligated stomach by oral gavage. Analysis of the dialysate samples was performed by HPLC-UV and concentration-time curves and pharmacokinetics analyses were used to determine differences between these tissue types.