A systematic statistical evaluation of five Folin–Ciocalteu (FC) assay protocols was performed to address the lack of standardization and limited reproducibility in total polyphenol content (TPC) determination. The protocols were critically compared using rigorous statistical analysis to identify the most accurate and reliable procedure. Method validation was carried out using a commercially available Vitaflavan extract and gallic acid standard solutions. The optimal protocol was found to involve a 120 min incubation with the FC reagent at room temperature, providing the highest accuracy and reproducibility among the tested approaches. The validated method was subsequently applied to determine TPC in extracts obtained from yellow onion skin bio-waste. Additionally, the effect of ultrasound-assisted extraction on TPC was investigated and compared with conventional solid–liquid extraction. The results demonstrated a significant increase in TPC when ultrasound treatment was applied, yielding 55.5 ± 1.3 mgGAE/g of dry onion skin compared to 32.9 ± 1.1 mgGAE/g obtained by conventional extraction. The proposed approach provides a statistically validated framework for improving the reliability and comparability of FC-based TPC measurements in complex matrices. Protocol P5 is recommended as a standardized, high-precision method for the reliable characterization of polyphenols in food-waste valorization and plant science.