Aim: to evaluate the anticancer effect of nano sized betanine particles (betanine NP) on squamous cell carcinoma cell line compared to doxorubicin (DOX) by measuring apoptosis through caspase 3. Material and Methods: Three groups of the human tongue squamous cell carcinoma cell line (SCC 25) were created, and two of them were treated with DOX and betanine NP. The third group served as a negative control and was not given any treatment. To evaluate the concentration of caspase 3 using the ElISA technology, different quantities of betanin NP and DOX were administered to SCC25 at 48- and 72-hour intervals. Results: Caspase 3 levels in the current investigation were 501.69.93, 543.86.71 pg/ml for DOX and 336.913.1, 405.53.5, and respectively for betanine NP in 48h and 72h intervals. Additionally, morphological analysis was performed to demonstrate the cells’ apoptotic alterations. There was a highly statistically significant difference between samples of various materials, as revealed by the ANOVA test for Comparison between groups using the ELISA technique for caspase 3 detection at 48h and 72h (p0.001). These findings suggest that DOX has a stronger apoptotic effect on SCC25 cells than betanin NP. Conclusion: Our findings explained that DOX and betanine NP can induce cancer cell death against SCC 25 cell lines by increasing the concentration of caspase 3. DOX has higher apoptotic effect on SCC25 cells than betanin NP according to ELISA technique.