The search for effective anticancer therapies has increasingly focused on natural compounds like Aloe vera, renowned for its therapeutic properties. This study investigates the anticancer properties of Aloe vera on 3D liver tumor spheroids via a PDMS-based microfluidic device, providing a more physiologically realistic model compared to traditional 2D cultures. HepG2 cells were cultivated to generate 3D spheroids on-chip, thereafter subjected to different concentrations of Aloe vera and the chemotherapeutic drug Doxorubicin to evaluate cytotoxic effects. The microfluidic system, validated by COMSOL simulations, facilitated continuous perfusion and real-time assessment of cell viability over a duration of 10 days. The results indicated that Aloe vera markedly diminished cell viability by triggering apoptosis at concentrations over 12.5 mg/mL. IC50 values were determined at 72 h: 25 ± 0.10 mg/mL for Aloe vera and 5.47 ± 0.03 µg/mL for Doxorubicin in 2D cultures, but in 3D cultures, the IC50 values were 31.25 ± 0.14 mg/mL for Aloe vera and 8.33 ± 0.05 µg/mL for Doxorubicin. This study underscores the promise of Aloe vera as a natural anticancer agent and illustrates the efficacy of microfluidic platforms for enhanced drug screening and customized medicine applications.