The electrophoretic mobility shift assay (EMSA) is a common technique to identify and analyze RNA-protein interactions, using the altered electrophoretic mobility of RNA and/or protein upon forming an RNA-protein complex. Traditional techniques of visualization of the EMSA results include either prelabeling of RNA before complex formation or specific RNA- or protein-staining after electrophoresis. Recently, two-color fluorescent staining (TCFS) methods were developed, in which the nucleic acid is stained first and scanned; subsequently, the protein is stained and scanned. In the current study, we developed a TCFS system, in which RNA and protein are stained with SYBR Green I and with SYPRO Red, respectively. The gel is subsequently scanned in two channels in a laser scanner to detect both simultaneously. Furthermore, we show that tetramethylrhodamine (TAMRA)-labeled proteins can subsequently be monitored in multicomponent RNA-protein complexes. This novel two-color fluorescence staining is simple, sensitive, and significantly faster than other comparable procedures and allows the independent quantitative determination of both free or complexed nucleic acids and proteins. The interactions between 23S rRNA and ribosomal protein L11 and the ribosomal protein complex L10/L12(4) were used to demonstrate the advantages of this method.