Maize GA21 line has integrated several tandemly repeated copies of the r-act 5-enol-pyruvylshikimate-3-phosphate synthase construct used for plant transformation. We were able to amplify a nucleotide sequence corresponding to the polylinker plasmid vector flanked by the r-act promoter and nopaline synthase 3′-terminator. A method for specific detection and quantification of Roundup Ready® transgenic maize line GA21 DNA using conventional and real-time PCR and based on this transgenic sequence is described. GA21 specific primers and probe were designed targeting the vector–promoter junction region and amplifying a 72-bp DNA fragment. Quantification methods were optimized through three different real-time PCR chemistries, i.e. SYBR® Green I, Amplifluor™ and TaqMan®. All three methods proved to be specific, highly sensitive and reliable for both identification and quantification of GA21 DNA. Plasmid pGAivr containing single copies of the GA21 and invertase amplicons was constructed for use as external standard in calibration curves. Using pGAivr, a TaqMan® based real-time PCR assay was optimized in duplex format targeting the maize species-specific ivr1 gene and the GA21 junction region. The detection limit of the method was 0.01% GA21, which is far below the established threshold for accidental presence of genetically modified organisms (GMO), this method therefore being suitable for use in routine GMO analysis.