Thyroid tumorigenesis involves qualitative and quantitative changes in protein expression, which can be comprehensively studied by proteome analysis. However, one of the technical bottlenecks of proteomics remains a reliable, sensitive and inexpensive method for quantification of differentially expressed proteins. This is due to the limited linear range of most available protein stains, i.e. silver and Coomassie blue, and high costs of commercially available fluorescent stains. In this paper we describe our experience with a lab-made ruthenium based fluorescent stain (ruthenium II tris(bathophenanthroline disulfonate) (RuBPs)) to perform proteome analysis of nodular thyroid disease. We first compared the properties of RuBPs with two highly sensitive protein stains: (1) silver staining and (2) the commercially available fluorescent dye Sypro Ruby. We show that in addition to its highly sensitive staining capabilities similar to Sypro Ruby and silver (2 ng), RuBPs offers several advantages such as a broad dynamic range (similar to Sypro Ruby and 500 times broader than the dynamic range of silver stain), low costs (€ 0.03 per gel) and excellent compatibility with mass spectrometry. We then applied the inexpensive RuBPs stain to 2D gels (pH 4–7) of four benign thyroid nodules and normal thyroid tissue. We were able to detect 1800 protein spots/gel in our thyroid samples. Quantitative changes in protein expression levels of at least 20–42 proteins were noted in the benign nodules compared with the normal thyroid tissue of the same patient. Differentially expressed spots were further characterised by nano-LC-FTICR and MALDI-TOF mass spectrometry. In summary we demonstrate, that the novel fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain is a highly sensitive, reliable and inexpensive tool for quantitative proteome analysis in thyroid nodular disease.