This study investigates the in vitro biological activities and computational predictions of pyrazolo[1,5-a]pyrimidine derivatives 12a and 12b. The compounds were synthesized and evaluated for their antioxidant, anti-diabetic, anti-Alzheimer's, anti-arthritic, and anticancer properties. In vitro assays showed that 12b exhibited higher antioxidant activity compared to 12a, with lower IC50 values against DPPH radicals and higher inhibitory activity against ABTS radicals. In anti-diabetic tests, 12b demonstrated significant inhibition of α-amylase, α-glucosidase, and β-glucosidase enzymes, with lower IC50 values than the standard drug acarbose. Electrophoretic analysis revealed that 12b altered the physiological state of α-amylase and denaturation of the protein portion in α-glucosidase, indicating potential anti-diabetic activity. In anti-Alzheimer's studies, 12b showed the highest inhibitory effect on acetylcholinesterase (AChE) activity, while in anti-arthritic tests, it exhibited the highest inhibitory effect on proteinase denaturation and activity. Cytotoxicity assays indicated that 12b had lower IC50 values against lung (A549) and colon (Caco-2) cancer cell lines compared to the standard drug doxorubicin, suggesting its potential as a cancer therapeutic. Computational predictions revealed that 12b is non-toxic and safe, with a higher therapeutic index than doxorubicin. The compounds also showed no carcinogenic, mutagenic, or immunotoxic effects. Overall, 12b demonstrated promising biological activities and safety, making it a potential candidate for further drug development.This study investigates the in vitro biological activities and computational predictions of pyrazolo[1,5-a]pyrimidine derivatives 12a and 12b. The compounds were synthesized and evaluated for their antioxidant, anti-diabetic, anti-Alzheimer's, anti-arthritic, and anticancer properties. In vitro assays showed that 12b exhibited higher antioxidant activity compared to 12a, with lower IC50 values against DPPH radicals and higher inhibitory activity against ABTS radicals. In anti-diabetic tests, 12b demonstrated significant inhibition of α-amylase, α-glucosidase, and β-glucosidase enzymes, with lower IC50 values than the standard drug acarbose. Electrophoretic analysis revealed that 12b altered the physiological state of α-amylase and denaturation of the protein portion in α-glucosidase, indicating potential anti-diabetic activity. In anti-Alzheimer's studies, 12b showed the highest inhibitory effect on acetylcholinesterase (AChE) activity, while in anti-arthritic tests, it exhibited the highest inhibitory effect on proteinase denaturation and activity. Cytotoxicity assays indicated that 12b had lower IC50 values against lung (A549) and colon (Caco-2) cancer cell lines compared to the standard drug doxorubicin, suggesting its potential as a cancer therapeutic. Computational predictions revealed that 12b is non-toxic and safe, with a higher therapeutic index than doxorubicin. The compounds also showed no carcinogenic, mutagenic, or immunotoxic effects. Overall, 12b demonstrated promising biological activities and safety, making it a potential candidate for further drug development.