Red maple leaf extracts (RME) were tested for their plant defense inducer (PDI) properties. Two extracts were obtained and compared by different approaches: RME1 using ethanol–water (30–70%, v/v, 0.5% HCl 1N) and RME2 using pure water. Both extracts titrated at 1.9 g L−1 in polyphenols and infiltrated into tobacco leaves efficiently induced hypersensitive reaction-like lesions with topical accumulation of auto-fluorescent compounds noted under UV and scopoletin titration assays. The antimicrobial marker PR1, β−1,3-glucanase PR2, chitinase PR3, and osmotin PR5 target genes were all upregulated in tobacco leaves following RME1 treatment. The alkaline hydrolysis of RME1 and RME2 combined with HPLC titration of gallic acid revealed that gallate functions were present in both extracts at levels comprised between 185 and 318 mg L−1. HPLC-HR-MS analyses and glucose assay identified four gallate derivatives consisting of a glucose core linked to 5, 6, 7, and 8 gallate groups. These four galloyl glucoses possessed around 46% of total gallate functions. Their higher concentration in RME suggested that they may contribute significantly to PDI activity. These findings define the friendly galloyl glucose as a PDI and highlight a relevant methodology for combining plant assays and chemistry process to their potential quantification in crude natural extracts.

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