Hidden insect infestation in stored rice grains, inflicted by Sitophilus oryzae and Sitophilus zeamais, continues to be a critical challenge to both producers and consumers across the globe and has far-reaching implications for food security, economic stability, and public health.These two genetically close insect species, commonly known as rice weevils, have long plagued rice storage facilities and are particularly adapted to attack rice grains and spend a considerable part of their life cycle, including the entire larval feeding period, inside them. They do this by creating an entrance hole, covering it after entry, and then, after the pupation process an exit hole is made, from which it then emerges, destroying rice grains.These insects constitute the hidden infestation that visual inspection cannot successfully detect, and traditional detection methods often fall short in reliably identifying them, necessitating the development of more robust and sensitive techniques, for precise identification of these insidious insect infestations.To address this pressing issue, this work focused on developing a specific and fast molecular detection method, such as a multiplex real-time polymerase chain reaction (RT-PCR). Firstly, adopting a DNA barcoding approach, by using a standardized region of insect genomes - the cytochrome oxidase I (COI) gene - was used as the foundation for species-specific primer and probe design and then their evaluation for efficiency on amplification of the target DNA. Secondly, an assay to determine the limit of detection (LOD) of the RT-PCR was done and later, the LOD of the technique itself, which is a critical parameter for its implementation as a quality control method in food processing and pest management in storage facilities. By doing so, this work offers a suitable tool for stakeholders seeking more effective means to detect, monitor, and combat hidden insect infestation in rice grains.