In the Czech Republic, Barley yellow dwarf virus (BYDV) and Wheat dwarf virus (WDV) are the prevailing viral species that can cause significant yield losses in cereal stands. Another virus, which has been on the increase in the Czech Republic and whose impact is expected to increase, is Wheat streak mosaic virus (WSMV). In this study, a TaqMan Multiplex Real-Time (qPCR) assay for simultaneous detection of BYDV (species PAV, PAS and MAV), WDV and WSMV was developed. Furthermore, additional assays for the viruses’ quantification in wheat and barley were developed. The assay was optimised and used to identify possible synergistic or antagonistic relations between the viruses in wheat and barley which would be characterised by increases or decreases of the titre of the individual viruses. For this, a greenhouse experiment with artificial inoculations of mixed and single infections of BYDV-PAV, WDV and WSMV was carried out, and field samples from the wheat and barley stands with mixed and single infections of BYDV and WDV were collected. No trend of increasing or decreasing titre was recorded for any of the viruses apart from WSMV titre which was decreased approximately five times in mixed infections with BYDV or WDV compared with the single WSMV infection. Further analyses are needed to expand the idea of possible interactions between WSMV and WDV or BYDV.