Flavescence dorée and Bois noir are two major grapevine yellows diseases affecting European viticulture. Accurate, early detection of the associated bacterial agents—Flavescence dorée phytoplasma (FDp) and ‘Candidatus Phytoplasma solani'—in both grapevines and insect vectors is critical for effective disease monitoring and control. Quantitative PCR (qPCR) is a highly sensitive molecular tool widely used for phytoplasma detection, often implemented in multiplex formats for simultaneous identification of multiple targets. However, the integration of internal quality controls is essential to validate DNA extraction efficiency and rule out PCR inhibition. While species-specific endogenous controls are commonly used, their limited host range restricts assay flexibility. This protocol describes a novel triplex qPCR assay enabling the simultaneous detection of FDp, ‘Ca. P. solani', and a universal endogenous control based on a conserved region of the eukaryotic 28S rRNA gene. This universal control allows broad applicability across diverse eukaryotic hosts, including both grapevine and key insect vectors, facilitating robust and flexible diagnostics in a single reaction setup.