High-throughput and non-destructive phenotyping approaches are increasingly needed to support precision agriculture and plant breeding. This study evaluates the use of unmanned aerial vehicle (UAV) multispectral imagery combined with machine learning to estimate anthocyanin content in red lettuce genotypes under field conditions. High-resolution RGB and multispectral images were acquired using a low-cost UAV platform, and vegetation indices sensitive to pigment variation were extracted at the plot scale. Ridge regression, decision tree, and random forest models were trained using 80% of the dataset and validated with the remaining 20%. Random forest achieved the highest performance for anthocyanin estimation, with coefficients of determination reaching R² = 0.84 and lower prediction errors than linear approaches. Overall, the results demonstrate that UAV-based multispectral sensing integrated with machine learning provides a robust, scalable, and cost-effective solution for non-destructive pigment phenotyping, with direct applications in biofortification-oriented breeding and precision agriculture.