Jets issuing through small holes in a wall into a freestream has proven effective in the control of flow separation. This technique is known as the vortex generator jet (VGJs) method. If a precursor signal of separation is found, the separation control system using VGJs can be operated just before the onset of separation and the flow field with no separation is always attained. In this study, we measured the flow field and the wall static pressure in a two-dimensional diffuser to find a precursor signal of flow separation. The streamwise velocity measurements were carried out in the separated shear layer and spectral analysis was applied to the velocity fluctuations at some angles with respect to the diffuser. The pattern of peaks in the spectral analysis changes as the divergence angle increases over the angle of which the whole separation occurs. This change in the spectral pattern is related to the enhancement of the growth of shear layer vortices and appears just before the onset of separation. Therefore, the growth of shear layer vortices can be regarded as a precursor signal to flow separation.