Lettuce is an important vegetable crop worldwide that widely grown hydroponically and usually consumed raw as salad. Developing an optimal harvest strategy could increase lettuce production and nutritional quality. The aim of the present research was to evaluate growth, mineral content, total phenols and flavonoids content and antioxidant activity of soilless (hydroponic) grown romaine lettuce (Lactuca sativa L. var. longifolia) leaves at three growth stages (“S1”-28 days after planting (DAP), “S2”-42 DAP, “S3”-56 DAP). Head fresh weight and number of leaves per head were higher at third growth stage; however, at this stage the size of lettuce head and leaf tissue hydration is highest which might affect the consumer choice for the produce. Regarding leaf chemical composition, the highest content of total phenols, flavonoids and antioxidant activity were observed during the first two growth stages. Mineral content of leaves generally decreased during plant development with significant highest contents being observed during the first growth stage for N and during the first two growth stages for P, K, Ca and Mg. In conclusion, the results of this study indicate that chemical composition of lettuce is highly dependent on plant development stage, and harvest stage might be considering for product biomass and bioactivity content to attain consumer’s acceptance.