Sesame (Sesamum indicum L.) is a herbaceous annual plant belonging to the family Pedaliacae. It is cultivated for its edible seed oil, which is an excellent source of balanced fatty acids. A sizable amount of the world’s edible oil is obtained from sesame. Phyllody is the most destructive disease of sesame and is caused by phytoplasma, a cell wall-less bacteria that is a phloem-harboring obligate pathogen. One of the distinctive features of this disease is the transformation of the floral organs into green leafy structures, which results in a loss of up to 80% of the seed output. Phytoplasmas are obligate intracellular parasites and are often present in very small numbers. As phytoplasmas are non-culturable, with unpredictable distribution, low concentration, seasonal variation, and enzyme-inhibitory plant polysaccharides and polyphenolic substances, they are frequently challenging to detect and identify. To lessen the economic loss due to phytoplasma infections, rapid, sensitive, accurate, and early detection is essential and might provide better scope to manage. Detection of phytoplasma through conventional methods such as microscopy, and protein-based assays is not sensitive and requires more expertise and resources. Traditional PCR methods offer some advantages and overcome the limitations of conventional methods to a great extent, but real-time polymerase chain reaction (qPCR) offers a number of benefits over normal PCR. It is a high-throughput, quick, sensitive, and reliable detection method. Hence, qPCR is crucial for the detection of phytoplasma as well as the analysis of host-pathogen interactions and epidemiological investigations. This chapter describes the procedures used in the qPCR assay for the identification of phytoplasma in both plants and insect vectors. To provide a holistic idea, the other methods have also been dealt with briefly.