With the method of the invention a wavelength-dependent refractive index of a specimen medium, which is examined with a light microscope, is determined. With the light microscope, a specimen measurement at the specimen medium which has an unknown refractive index is performed, wherein illumination light is radiated to the specimen medium and detection light coming from the specimen medium is measured. With the specimen measurement, a specimen measurement focus position of the illumination and/or detection light is measured. Using a mathematical model, in which a focus position of illumination and/or detection light is defined in dependence of a refractive index of a medium, the refractive index of the specimen medium is derived from the specimen measurement focus position. Furthermore, a light microscope for carrying out the method is described.