Short-chain fatty acids (SCFAs) are the main metabolites produced by bacterial fermentation of non-digestible carbohydrates in the gastrointestinal tract. They can be seen as the major flow of carbon from the diet, through the microbiome to the host. SCFAs have been reported as important molecules responsible for the regulation of intestinal homeostasis. Moreover, these molecules have a significant impact on the immune system and are able to affect inflammation, cardiovascular diseases, diabetes type II, or oncological diseases. For this purpose, SCFAs could be used as putative biomarkers of various diseases, including cancer. A potential diagnostic value may be offered by analyzing SCFAs with the use of advanced analytical approaches such as gas chromatography (GC), liquid chromatography (LC), or capillary electrophoresis (CE) coupled with mass spectrometry (MS). The presented review summarizes the importance of analyzing SCFAs from clinical and analytical perspective. Current advances in the analysis of SCFAs focused on sample pretreatment, separation strategy, and detection methods are highlighted. Additionally, it also shows potential areas for the development of future diagnostic tools in oncology and other varieties of diseases based on targeted metabolite profiling.