Areca nut, commonly known as betel nut, holds significant cultural importance in Asian societies. However, its aflatoxin associated losses are more common in Pakistan, Nepal, India and Bangladesh. This study aimed to optimize total aflatoxin determination in areca nut using cost effective, accurate and valid techniques. Various strategic modifications were applied during the method optimization. Blending an 80:20 methanol: water mixture with areca nut sample at high speed, followed by dilution with 15% Tween 20, was found to yield reliable and repeatable results. Validation results aligned with the EU Directive 2002/657/EC and AOAC standard validation guidelines, with % RSD ˂ 20-21% and recovery rates between 60 – 110 %. Detection and quantification limits were low, indicating method sensitivity. Methods were found rugged but showed cross reactivity against tannin and phenolic compounds. Measurement uncertainty was ± 5.8µg/kg for fluorometry and ± 1.9 µg/kg for ELISA. Statistical comparison using ANOVA against the HPLC-FLD reference method revealed no significant differences (p = 0.991), confirming result consistency and homogeneity of variance (Levene’s statistic = 0.009). In conclusion, optimized fluorometry and ELISA methods for aflatoxin analyses in areca nut are cost-effective, reproducible, sensitive, and statistically comparable to the reference method, making them ideal for routine analysis.