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Phytohormones are important plant components that are involved in a signaling cascade in plant development. In strawberry, the influence of abscisic acid (ABA) and its different forms [phaseic acid (PA), dihydrophaseic acid (DPA), and ABA glucose ester (ABA-GE)] on the process of fruit maturation is not yet completely understood. Quantification of phytohormones is currently performed by liquid chromatography coupled to mass spectrometry (LC-MS) due to its sensitivity and specificity. However, the sample matrix and the extraction procedure will influence the analysis. Thus, this study aimed to optimize a simple extraction method and validate the LC-MS quantification of ABA as well as the identification and quantification of ABA derivatives (PA, DPA, and ABA-GE) in strawberry fruit. Hormone extraction was performed using either methanol (80% v/v—S1) or acetone:water:acetic acid (80:19:1 v/v—S2) solutions with or without the use of sonication. The most efficient extraction was obtained using S1 without sonication, and LC-MS validation parameters for ABA were within acceptable scores.