•The design of the experiment enabled the study the effects of UV and temperature on phenolics.•UV radiation stimulated accumulation of flavonoids, anthocyanins and methoxycinnamic acid.•Temperature was a major factor influencing concentrations of rosmarinic, p-anisic and vanillic acid.•The non-invasive fluorescence method can be used to estimate flavonoid content in lettuce.•The trends of phenolic compound accumulation in green and red genotypes were similar. Environmental conditions influence the content and metabolic profile of phenolic compounds in vegetables. The present study focused on distinguishing between the effects of ultraviolet (UV) radiation and temperature on lettuce plants with green or red leaf color when grown in either a greenhouse or outdoors. A combination of the non-destructive, fluorescence-based method with spectrophotometric and HPLC analyses enabled us to assess the effects of environment, cultivar, and plant-leaf color on concentrations of flavonoids, anthocyanins, and phenolic compounds. The accumulation of total phenolics, flavonoids, anthocyanins, and phenolic acids (benzoic acid derivatives and cinnamic acid derivatives) increased in direct sunlight (high UV radiation, moderate temperature) conditions outdoors as compared to the greenhouse conditions (low UV radiation, high temperature). The comparison of the effects of UV radiation and temperature (indoor-outdoor effect) indicated that the level of UV radiation plays a dominant role in the accumulation of flavonoids, anthocyanins, and methoxycinnamic acid; while temperature predominantly influences the accumulation of phenolic acids (rosmarinic, p-anisic, vanillic acid. Although, the leaf color (green vs. red) was strongly related to the content of majority of studied phytochemicals, environmental conditions affected their concentrations in both color types similarly. The concentrations of compounds estimated with the non-invasive, fluorescence excitation ratio method were highly consistent with those obtained by standard analytical approaches. Our results show that this fast, non-invasive method can be effectively used for determining concentrations of flavonoids and phenolic acids in lettuce plants.