Recently, a new type of synthetic marijuana called “American grass” (“Co My” in Vietnamese), a fast-acting and strong-addictive illicit drug, has been increasing used in numerous criminal and law-breaking actions in Vietnam. One of the prominent compounds commonly detected in American grass is the synthetic cannabinoid 5-Fluoro-MDMB-PICA. However, limited information have been reported on the extraction and analysis of this compound in American grass. Therefore, this study, for the first time, extracted, isolated, structural elucidated, and gas chromatography/mass spectrometry (GC/MS) quantitated this compound from American grass samples. To this end, the 5-Fluoro-MDMB-PICA was successfully extracted by simple sonication-assisted maceration method with dichloromethane, isolated by fractional column chromatography with n-hexane:ethyl acetate (6:4 v/v), purified by re-crystallization, purity-checked by high performance liquid chromatography (HPLC), and structural elucidated by spectral methods of UV–Vis, infrared (IR), MS, and nuclear magnetic resonance (NMR). Then, the GC/MS quantification method for 5-Fluoro-MDMB-PICA was developed, with a fast running time of 7 min, and fully validated following ICH guideline, which met all the requirements of specificity, linearity (y = 0.0380x - 0.0295; R2 = 0.9986), range (9.954 µg/mL to 99.540 µg/mL), accuracy (recovery rate of 100.45 ± 0.62%), intermediate precision (RSD% of 1.92 ± 0.05%), limit of detection (0.06 µg/mL), and limit of quantitation (0.2 μg/mL). Finally, the GC/MS method was utilized for the quantification of 5-Fluoro-MDMB-PICA in 20 American grass samples, which demonstrated that the compound average concentration was 2.2% w/w. Conclusively, these results contribute to the present literature gap on the analytical method for 5-Fluoro-MDMB-PICA in American grass.