Bile acids (BAs) are traditionally recognised as detergents that facilitate lipid and glucose digestion and homeostasis. More recently, they have emerged as signalling molecules that can influence metabolic processes via the gut-brain axis. BAs are implicated in a wide range of diseases and conditions, including hepatobiliary, metabolic, cardiovascular, neurological disorders, and gastrointestinal disorders, such as irritable bowel syndrome (IBS). This research aims to develop an analytical method capable of resolving isomeric BA species (compounds with identical molecular weights but distinct structural configurations) using a single chromatographic column and utilising a liquid chromatography tandem mass spectrometry (LC-MS/MS) instrument. Ultimately, this method is being developed to compare BA profiles between individuals with IBS and healthy controls, to identify BAs as potential biomarkers for IBS. This study will discuss recent developments and milestones achieved through various decision-making processes, including the selection of columns and mobile phases, as well as the procedures for blood collection required for this study. Most importantly, it will be showcased that in complex metabolites, such as BAs, the molecular weight tends to be a recessive trait, often portraying chameleonism, and that isomerism continues to present significant challenges in this modern analytical world.