Infrared (IR) spectroscopy is a vibrational spectroscopic technique based on the absorption of infrared radiation by matters that excite vibrations of molecular bonds. It is a powerful method for investigating structural, functional, and compositional changes in biomolecules, cells, and tissues. In recent years, scientific researchers have continued to increase the performance of this technique on clinical cases such as cancers and metabolic disorders. Obesity is one of the main factors that increases the risk of many diseases and contributes to functional disabilities in tissues such as adipose, liver, and muscle. Applications of IR spectroscopic techniques allow identifying molecular changes due to obesity, to understand the molecular mechanism of the disease, to identify specific spectral biomarkers that can be used in diagnosis. In addition, these spectral biomarkers can be used to identify the appropriate drugs and their doses for treatment. In this chapter, applications of IR spectroscopic and microscopic techniques to the characterization and understanding the obesity metabolism will be presented. The discriminatory power of these techniques in diagnosis of obesity will be discussed. In future, these novel approaches will shed light on the internal diagnosis of obesity in clinical application.