A new promising treatment for patients with symptomatic cholelithiasis is extracorporeal shock wave lithotripsy. In order to learn how to interpret the computed tomographic (CT) densities of gallstones measured in vivo, our radiologists asked the clinical chemistry laboratory to analyse cholesterol, bilirubin and calcium in gallstones. No reference methods for the analysis of gallstones have been described. A literature search for manageable quantitative methods for these analyses was not successful. Among the different X-ray diffraction, infrared and chemical analyses described, we could find no well documented analyses that have been compared with reference methods or proposed reference methods for serum. This finding prompted us to develop chemical methods for cholesterol, bilirubin and total calcium in gallstones starting from (proposed) reference methods for serum and to investigate the accuracy, imprecision and linearity of these methods.