Mycoplasma contamination of a known cell line is detected by collecting a Raman spectrum of a targeted volume within a sample, the targeted volume containing a known cell line of interest, obtaining a reference spectrum uniquely associated with the known cell line where the obtained reference spectrum is known to be free of mycoplasma and comparing, using a processing device, the reference spectrum to the collected spectrum. Mycoplasma is further detected by identifying whether there are unnatural molecular compositions within the collected spectrum based upon the comparison of the reference spectrum to the collected spectrum and providing an indication as to whether mycoplasma is detected in the collected Raman spectrum based upon whether unnatural molecular compositions are identified within the collected spectrum.
