In this chapter, the authors introduce a technology that allows vibrational imaging of chemical content in a deep tissue through photoacoustic detection of vibrational overtone and combination absorption of molecules. Optical imaging of biological tissue presents a number of advantages, including high resolution, noninvasive to tissue, label free, and chemical composition selectivity. Vibrational photoacoustic imaging (VPAI), or vibrational photoacoustic tomography, which inherits all the advantages of PAI, can provide chemical and molecular information by spectroscopically detecting the vibrational absorption happened to the specific chemical bond through overtone or combination transitions. Optical absorption in VPAI is based on the vibrational overtone or combination transitions. Multispectral (MS)-PAI is a powerful modality that allows for highly specific chemical mapping in a biological tissue through MS/multiwavelength scanning of the excitation laser across the characteristic absorption spectral region of interested molecules. Among the quantitative analysis methods of MS-PAI, the most widely used is multivariate curve resolution alternating least squares method.