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The use of nanoparticles in nanomedicine has received increasing interest. However, in vivo detection of nanoparticles using optical techniques is still a formidable challenge. Detecting surface‐enhanced Raman scattering (SERS)‐labeled nanoparticles through thick tissue is crucial due to its large number of potential applications in the field of disease diagnostics and monitoring. As gold nanoparticles are becoming an important nanoprobe and nanosensor platform for SERS in vivo applications, accurate detection and quantitation of these particles has become even more important. Conventional optical methods, however, are typically limited in obtaining SERS signals at the surface level, due to the attenuation caused by the highly scattering and absorbing tissue. Herein, we utilize spatially offset Raman spectroscopy to overcome this depth limitation and obtain specific spectrochemical signatures of SERS …