A liquid chromatographic method for the determination of methylene blue and leucomethylene blue in male and female Fischer 344 rat urine and male and female B6C3F1 mouse urine was developed for use in supporting toxicokinetic studies, validated, and used to analyze urine samples for a preliminary dose level range-finding study. The method was validated for a concentration range of 10.0 to 20,000 ng/mL in urine. Samples up to 75,000 ng/mL demonstrated good recoveries when diluted into the range of the calibration curve. Six sets of calibration standards were prepared in F344 male rat urine for analysis to demonstrate reproducibility and ruggedness. The stability of sample extracts was determined under various storage conditions. During the course of analyses of the animal samples, a possible metabolic process was observed. Although Azure B is a significant impurity in methylene blue trihydrate, the amount of Azure B seen in urine samples collected from rodents dosed with methylene blue trihydrate is significantly greater than the amount seen in rodent urine spiked directly with methylene blue. This observation suggests possible N-demethylation of the methylene blue as a metabolic transformation to Azure B.