Since the publication of our first analytical method in 2000 to detect and quantify phthalate metabolites in human urine, we have modified the method several times to improve performance, reduce the volume of matrix and solvents used, and to increase the number of analytes in one analytical run. We performed cross method validation and ruggedness testing after each modification to ensure that the analytical method adopted is robust and produces accurate and reproducible data when compared to the previously used method. Here, we present the results from the evaluation of the ruggedness of our analytical approach under variable experimental conditions, using the current analytical method. Minor deviations of the standard experimental conditions, i.e., pH, incubation time, amount of deconjugation enzyme, and incubation temperature, had no effect on final analyte concentrations. Furthermore, we validated the method to ensure accuracy at concentrations beyond the highest calibration standard. The concentrations obtained by using a lower volume of urine agreed well with original levels, suggesting broad linear calibration range as well as complete hydrolysis of the glucuronide conjugates with the standard amount of beta-glucuronidase used for deglucuronidation; also, the time of incubation (90 min) was adequate regardless of the amount of glucuronide present. We also summarize the precision of concentration data acquired by the five different analytical approaches we have used since 2000. The correlation plots of concentration data for each analyte obtained from split sample analysis, using three of these approaches, produced linear curves (R(2)>0.98) with slopes and intercepts that were not statistically different (p>0.05) from 1 and 0, respectively. These results suggest that the data are reproducible and accurate, regardless of the analytical method used. Furthermore, analysis of quality control urine samples made over the years confirmed the stability of the phthalate metabolites in urine at -70 degrees C for several years and the consistency of the analytical measurements obtained by using various methodological approaches over time.