Raman spectroscopy has been well established as a powerful in vitro method for studying biological tissue and diagnosing disease. The recent development of efficient, high-throughput, low-background optical fiber Raman probes provides, for the first time, the opportunity to obtain real-time performance in the clinic. We present an instrument for in vivo tissue analysis which is capable of collecting and processing Raman spectra in less than 2 s. This is the first demonstration that data acquisition, analysis, and diagnostics can be performed in clinically relevant times. The instrument is designed to work with the new Raman probes and includes custom written LabVIEW and Matlab programs to provide accurate spectral calibration, analysis, and diagnosis along with important safety features related to laser exposure. The real-time capabilities of the system were demonstrated in vivo during femoral bypass and breast lumpectomy surgeries. Such a system will greatly facilitate the adoption of Raman spectroscopy into clinical research and practice. © 2005 Society of Photo-Optical Instrumentation Engineers.