Abstract: The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is an important pest of the Americas. This insect has a very wide host range and has been able to attack several crops; however, the most frequently hosts are maize and sorghum causing an economic impact. Alphabaculoviruses (lepidopteran nucleopolyhedroviruses, NPV) and betabaculoviruses (granuloviruses, GV) of the family Baculoviridae have been isolated causing co-infection in fall armyworm populations in Colombia. This condition has a considerable potential as biological control strategy, as in some cases GVs are able to enhance the infectivity and virulence of NPVs. In this work a sensitive tool for detection and quantitation of NPVs and GVs in mixed infections in S. frugiperda was developed. We used a multiplex real-time PCR (q-PCR) method based on highly specific oligonucleotides and Taq-man probes that recognize fragments of polyhedrin (polh) and granulin (gran) genes. The specificity of individual probes showed non-cross amplification between NPVs and GVs. Besides, each probe was able to detect NPVs or GVs from different geographical origins. Samples of mixed SfMNPV/SfGV genomes at different ratios were analysed and interferences were not observed. The minimum detection limit of the technique was 6.4 x 10-4 ng /μl of DNA, equivalent to 1.25 x 103 gene copies (gran and polh) and minimal variation inter-assays showed that the technique was reproducible. This technique is essential in the process of biopesticide developing based on alpha- and betabaculoviruses, including the detection of larvae infected in field until the quality control of final formulated product.