Stimulated Raman scattering (SRS) microscopy, based on vibrational spectroscopy, is able to perform label-free imaging with high sensitivity, high spatial and spectral resolution, 3D sectioning, and fast time of image acquisition, ie, a few seconds. In this chapter, the implementation of a femtosecond SRS microscope, working in C▬ H or O▬ H region (> 2800 cm− 1) of Raman spectra of biomolecules, is reported. Our microscope is realized by integration of a femtosecond (fs) SRS experimental setup with an inverted optical microscope equipped with fast mirror scanning unit. The microscope is provided with two femtosecond laser sources: a titanium-sapphire (Ti: Sa) laser oscillator and an optical parametric oscillator (OPO). In addition, the reliability of our system in C▬ H region is tested by localizations of lipid droplets inside adipocyte cells. Finally, the extension of microscope to silent region of< 1800 cm− 1 and fingerprint region of 1800–2800 cm− 1 is also achieved by adding a second-harmonic generator to cascade the OPO and double its energy radiation. Definitely, our microscope is able to take on some recent challenges of SRS microscopy, including improvements of sensitivity and detection specificity.