Plant biochemistry studies have increased in recent years due to their potential to improve human health. Argylia radiata is an extremophile plant with an interesting polyphenolic profile. However, its biomass is scarce and occasionally available. Argylia in vitro biomass was obtained from tissue culture and compared with in vivo roots regarding its polyphenolic and flavonoid content. Different solvents were used to prepare extracts from the in vitro tissue of callus and aerial plant organs and in vivo roots. UPLC-MS/MS was used to assess the chemical composition of each extract. ORAC-FL and scavenging of free radicals (DPPH and OH) methods were used to determine the antioxidant capacity of extracts. Furthermore, the biological activity of the extracts was established using the cellular antioxidant activity method. The vitroplants were a good source of polyphenols (25–68 mg GAE/100 g tissue FW), and methanol was the most efficient solvent. Eight polyphenolic compounds were identified, and their antioxidant properties were investigated by different chemical methods with EPR demonstrating its specific scavenging activity against free radicals. All extracts showed cellular dose-dependent antioxidant activity. The methanolic extract of vitroplants showed the highest cellular antioxidant activity (44.6% and 51%) at 1 and 10 g/mL of extract, respectively. Vitroplants of A. radiata are proposed as a biotechnological product as a source of antioxidant compounds with multiple applications.