Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology. About 131 spots were recognized as phosphorylated proteins as they showed different migration behaviour after phosphatase treatment. The analysis of 42 selected spots was carried out by LC/MS/MS technology resulting in the identification of two new phosphoproteins.