Loose smut of barley, caused by fungal pathogen Ustilago nuda is one of the major concerns throughout the globe for barley producers. The infection takes place without exhibiting any obvious symptoms and an infected seed lot can only be identified at the heading stage when the fungal teliospores emerge at the place of crop. The percentage losses on yield are directly proportional to the occurrence of infection. Currently available detection methods include seed health testing protocols which are time-consuming and cumbersome. With the globalization of the international market and increased crop demand, development of rapid disease screening methodologies has become an essential focus in the field of plant pathology. The present study sought to develop a rapid probe-based detection method for screening of U. nuda with real-time qPCR. Two U. nuda specific primer pairs were compared using standard PCR alongside optimization of real-time qPCR assay. The advantage of high fidelity DNA polymerase for amplification of U. nuda genomic DNA was recorded. U. nuda genomic DNA was amplified and cloned into a vector which was further used for generation of a quantification curve with a specific probe. The qPCR assay developed in this study was successful in the detection of as little as 43 copies of U. nuda genomic DNA. With studies involving larger sample size and field samples, this assay can be improved for enhanced sensitivity and specificity which can help in monitoring infection from DNA extractions of barley seeds and further improving the current microscopic detection methods.