Improved methods are needed for in situ characterization of post-translational modifications (PTMs) in cell lines and tissues. It is, for example, desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors, to evaluate therapeutic responses. Unfortunately, the leading methods for observing the dynamics of tissue PTMs in situ – immunohistochemistry and immunofluorescence – exhibit limited sensitivity and selectivity. Proximity ligation assay (PLA) is a novel method which offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA-amplification as a component of detection of the target molecule. Here we therefore applied a generalized in situ PLA to investigate phosphorylation of platelet derived growth factor receptor ß (PDGFRß) in cells stimulated with PDGF-BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands, which was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRß in human embryonic kidney cells stably over-expressing HA-tagged human PDGFRß, in porcine aortic endothelial cells transfected with the ß-receptor, but not in cells transfected with the a-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRß. We furthermore visualized tyrosine phosphorylated PDGFRß in tissue sections from fresh-frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents and to enhance the diagnostic potential in histopathology.