Phytoplasmas are plant pathogenic bacteria that have been associated with disease in over 1000 different plant species. In Papua New Guinea (PNG), crops of economic importance that are affected by banana wilt associated phytoplasma (BWAP) include coconut, betelnut and banana plants. Reliable detection of phytoplasma is important for preventing spread to unaffected areas and as part of management practices for crops where phytoplasma has been associated with disease. BWAP is not currently present in Australia, and it is unknown whether BWAP poses a risk to Australian-produced dessert banana production or to other plants. Preventing incursions of BWAP is therefore imperative to protecting commercial banana production in Australia. In general, detection of phytoplasma is challenged by the low titre of phytoplasmas in some hosts, which can result in false negatives. Molecular assays that are reported for universal phytoplasma detection are prone to false positives due to the similarity of commonly targeted genomic regions of phytoplasmas to those of bacterial endophytes in banana plants. Several diagnostic qPCR assays have been designed for specific phytoplasma detection but none have been developed for the targeted detection of BWAP. This study sought to validate a novel qPCR assay that was designed for detection of BWAP in banana tissues. Validation was conducted by measuring assay performance against parameters such as specificity, sensitivity, repeatability and robustness. Validation of a qPCR assay with low false positive rates and enhanced sensitivity to BWAP improves result reliability. This validation supports its use as a first-line detection tool for BWAP, strengthens diagnostic capabilities, and facilitates the control and management of phytoplasma associated wilt in banana and coconut.