Metabolomics investigates small molecules in biological fluids to understand changes during exposure to factors such as environmental agents or diseases. Liquid chromatography hyphenated with mass spectrometry (LC–MS) is widely used to detect biochemical alterations in pathophysiological processes. Targeted metabolomics focuses on specific compound classes or pathways, providing the high sensitivity and quantification required to translate findings into clinically relevant concentrations. In bioanalysis, LC–MS encounters challenges when quantifying endogenous metabolites due to the absence of a blank matrix for external calibration (EC). This thesis explores the fundamentals of LC–MS analytical calibration and current quantification strategies from both theoretical and practical perspectives. Internal calibration (IC) was evaluated as an alternative to EC, using stable isotope-labeled standards for straightforward analyte concentration determination. The IC approach was successfully implemented to quantify endogenous steroids and metabolites relevant to chronic kidney disease progression. This methodology demonstrated accuracy comparable to traditional EC-based quantification, underscoring its value for clinical metabolomics in a hospital setting.