Despite the growing interest in phytoecdysteroids due to their valuable biological effects, there remains a notable gap in accessible methodologies for their reliable identification and quantification in plant-derived products. Among these compounds, ecdysterone has attracted substantial attention owing to its broad pharmacological activity and proposed performance-enhancing effects. Similarly, turkesterone has emerged as a promising candidate within the domain of sports supplementation, given its hypothesized anabolic potential. This study aimed to develop and validate a robust liquid chromatography–mass spectrometry (LC–MS) method for the qualitative and quantitative analysis of ecdysterone and turkesterone. Method optimization focused on ionization efficiency and chromatographic resolution using a mobile phase of 0.1% formic acid in water and acetonitrile. An analysis was conducted in both total ion chromatogram and selected ion monitoring modes for accurate detection and quantification. The method was systematically optimized to ensure high selectivity, sensitivity, and reproducibility while maintaining cost-effectiveness to support its implementation in routine phytochemical screening and quality control applications. The validation results confirmed the method’s analytical reliability, demonstrating excellent linearity, accuracy, precision, and detection limits. The practical applicability of the method was further established through the analysis of plant extracts. Among the evaluated plant extracts, kaniwa exhibited the highest concentration of ecdysterone, followed by spinach, quinoa, and asparagus. These findings emphasize the nutritional value of select plant-based foods as natural sources of phytoecdysteroids and demonstrate the utility of the developed LC–MS method for quality control in sports supplements and natural product research.