Enzyme-linked immuno-sorbent assay (ELISA) is widely used for biomarker detection. A good biomarker can distinguish patients from healthy or benign diseases. However, the ELISA method is not suitable for small molecule or trace substance detection. Along with the development of new technologies, an increasing level of biomaterials, especially small molecules, will be identified as novel biomarkers. Quantitative immuno-PCR, chromatography-mass spectrometry, and nucleic acid aptamer are emerging methodologies for detection of small molecule biomarkers, even in living cells. In this review, we focus on these novel technologies and their potential for small molecule biomarkers and living cell analysis.