A simple, sensitive, and rapid assay based on liquid chromatography coupled to tandem mass spectrometry was designed for simultaneous quantitation of secondary metabolites in order to investigate the influence of variety and agronomic conditions on the biosynthesis of bioactive compounds in strawberry. For this purpose, strawberries belonging to three varieties with different sensitivity to environmental conditions ('Camarosa', 'Festival', 'Palomar') were grown in a soilless system under multiple agronomic conditions (electrical conductivity, substrate type, and coverage). Targeted metabolomic analysis of polyphenolic compounds, combined with advanced chemometric methods based on learning machines, revealed significant differences in multiple bioactives, such as chlorogenic acid, ellagic acid rhamnoside, sanguiin H10, quercetin 3-O-glucuronide, catechin, procyanidin B2, pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside, and pelargonidin 3-O-rutinoside, which play a pivotal role in organoleptic properties and beneficial healthy effects of these polyphenol-rich foods.