Early plant selection for desirable traits is important in tree improvement programs and sustainable forest management. In this study, we demonstrate the use of image based high-throughput plant phenotyping (HTPP, LemnaTec 3D Scanalyzer, Germany), with Red, Green, Blue (RGB), and hyperspectral cameras, to quantify Quercus bicolor and Quercus prinoides seedlings growth and development [plant height, projected leaf area (LA), plant/canopy width, ConvexHull, and plant aspect ratio], and assess their response to a dry-down period, under controlled environment. HTPP images were validated against low throughput measurements, including gas exchange, leaf spectral properties, and morphological traits. Using HTPP, we recorded significant differences in growth dynamic in examined species, with faster initial growth rate early in the growing season, higher photosynthetic rates, larger LA, and seedling dimension, in Q. bicolor, compared to Q. prinoides. This has ecological implications on species responses to shading and timing of drought stress, as well as their competitive relationship with each other and with other species under changing climate. HTTP showed that both growth and leaf expansion ceased under dry-down treatment. Image derived and measured morphological traits were highly and significantly correlated under both well-watered and dry-down conditions for both species. To obtain meaningful physiological information using spectrometry, we calculated 12 vegetation indices (VIs) from both HTPP and handheld spectrometers. Vogelmann and Maccioni indices had the highest correlations across methods, suggesting their potential use for assessing oak seedlings performance and health. Our results emphasized the importance of VIs ground truthing, since VIs performance can vary significantly between species and treatments. HTPP tools can successfully be used to effectively assess forest seedlings of the two Quercus species, important for early plant selection for forest management purposes and tree improvement programs.