Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we have demonstrated that Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we have applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by BDNF (brain derived neurotrophic factor), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitates (pY IPs) comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase (RTK) for BDNF, and others such as Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are proteins known to regulate intracellular trafficking of RTKs. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics.