Measuring bile acids in feces has an important role in disease prevention, diagnosis, treatment, and can be considered a measure of health status. Therefore, the primary aim was to develop a sensitive, robust, and high throughput liquid chromatography tandem mass spectrometry method with minimal sample preparation for quantitative determination of bile acids in human feces applicable to large cohorts. Due to the chemical diversity of bile acids, their wide concentration range in feces, and the complexity of feces itself, developing a sensitive and selective analytical method for bile acids is challenging. A simple extraction method using methanol suitable for subsequent quantification by liquid chromatography tandem mass spectrometry has been reported in, "Extraction and quantitative determination of bile acids in feces" [1]. The data highlight the importance of optimization of the extraction procedure and the stability of the bile acids in feces post-extraction and prior to analysis and after several freeze-thaw cycles.