This research investigates the impact of various concentrations and types of polyamines, as well as diverse explant sources including flower buds and fruits, on callus formation, plant regeneration, and phenolic compound content of the medicinally significant caper plant. Different types, concentrations of polyamines were used in MS culture medium. Experimental results showed that polyamine varieties and concentrations increased callus induction and regeneration and affected the accumulation of quercetin and rutin. The highest callus formation was observed in 0.5 mg/l spermine cultures in flower bud explants and 1.0 mg/l putrescine cultures in fruit explants. Plant regeneration was highest at 1.0 mg/l putrescine application in flower buds, 0.5 mg/l spermine and spermidine in fruits and was not seen in either of the control groups. Phytochemical analysis showed higher production of quercetin (flower buds; 1.3 μg/g and fruits; 0.6 μg/g) and rutin (flower buds; 1.9 μg/g and fruits; 0.9 μg/g), in callus cultures in the presence of 0.5 mg/l spermidine, 1.0 mg/l putrescine, 0.5 mg/l putrescine, 0.5 mg/l spermidine, respectively. This study reveals that polyamines can be effectively used for the enhancement of callus formation, plant regeneration and secondary metabolites in the callus culture of highly medicinal plant, caper. Hence, these discoveries have the potential to be applied in the industrial-scale cultivation of caper plants and the production of pharmacologically active metabolites.