Urbanization has provided greater demand for food, and the search for strategies capable of reducing waste is essential to ensure food security. Lettuce (Lactuca sativa L.) culture has a short life cycle and its harvest point is determined visually, causing waste and important losses. Using vegetation indices could be an important alternative to reduce errors during harvest definition. The objective of this study was to evaluate different vegetation indices to predict the growth rate and harvest point of lettuce. Twenty-five genotypes of biofortified green lettuce were evaluated. The Green Leaf Index (GLI), Normalized Green Red Difference Index (NGRDI), Spectral Slope Saturation Index (SI), and Overall Hue Index (HUE) were calculated from images captured at 1, 8, 18, 24, and 36 days after transplanting (vegetative state). The diameter and average leaf area of plants were measured using QGIS software. Green mass, number of leaves, and plant and stem diameter were measured in the field. The means were compared using the Scott–Knott test (p ≤ 0.05) and simple linear regression models were generated to monitor the growth rate, obtaining R2 values ranging from 62% to 99%. Genetic dissimilarity was confirmed by the multivariate analysis presenting a cophenetic correlation coefficient of 88.49%. Furthermore, validation between data collected in the field versus data obtained by imaging was performed using Pearson’s correlations and showed moderate to high values. Overall, the vegetation indices SI, GLI, and NGRDI were efficient for monitoring the growth rate and determining the harvest point of different green lettuce genotypes, in attempts to reduce waste and losses. It is suggested that the definition of the harvest point based on vegetation indices are specific for each genotype.