Some groups have been reported after annealing graphene on SiO2/Si substrate, the value of I(2D)/I(G) decreases. So on the basis of intensity ratio of Raman peaks, after annealing, we can not say graphene is mono layer or bi-layer.
The SAED pattern can be used to distinguish between single and few layer Graphene. by analyzing the intensity profile plotted through diffraction spots. It is reported that if the inner reflections are intense as compared to the outer reflection, which indicate that the sample is single layer. On the other hand for if the outer reflections are intense than outer reflections peaks it is a few layer graphene.
Please see the following articles.
Y. Hernandez, V. Nicolosi, M. Lotya, F. M. Blighe, Z. Sun, S. De, I. T. Mcgovern, B. Holland, M. Byrne, Y. K. Gunˈko, J. J. Boland, P. Niraj, G. Duesberg, S. Krishnamurthy, R. Goodhue, J. Hutchison, V. Scardaci, A. C. Ferrari and J. N. Coleman, Nat. Nanotechnol. 2008, 3, 563-568.
H. Tang, G. J. Ehlert, Y. Lin and H. A. Sodano, Nano Lett., 2011, 12, 84–90.
Other than the Raman and SAED, BET surface area also gives idea about your graphene. calculated value of specific surface area for monolayer graphene is 2630 m2g-1 it reduces when the layers are stacked.
In Raman the peak shape of 2D band gives a clear difference of mono and bilayer graphene. Try lorentian peak fitting. The bilayer 2D peak is composed of sub bands.
Generally, layer number of graphene does not change by post annealing. There may be some change in the 2D peak position/Intensity due to other factors. Please go through the following article for more information.
After annealing of graphene, Intensity ratio I(2D)/I(G) decreases and FWHM of 2D peak increases due to some defects or may be due to strain. So I think Lorentzian fitting of 2D peak will not help in this case.