Amino acids are naturally occurring compounds in many edible or medicinal plants, which possess a variety of nutraceutical effects in human. Here, a method of ultrasound-assisted extraction was optimized using Box–Behnken design of response surface methodology in maximizing the yield of free amino acids deriving from flowers of Apocynum venetum L. Under the optimal condition of ultrasound-assisted extraction, i.e. 187.5 W of ultrasonic power, 31.93 min of extraction time, and 0.47 mg/ml of solid–liquid ratio, the experimental yield of extractive was 287.17 ± 0.205 mg/g, which was in close agreement with the value, as predicted by the established model. In addition, a hydrophilic interaction ultra-high performance liquid chromatography coupled with QqQ-MS/MS method was developed and validated for simultaneous quantification of 20 types of amino acids without derivatization contained in A. venetum flowers. The analytical method was validated by matrix effect, linearity, limit of detection, limit of quantification, precision, repeatability, stability, and recovery. The analysis results showed that A. venetum flower was rich in free amino acids of ~3% of total dried weight, and which contained leucine (13.7 g/mg), isoleucine (7.9 g/mg), and lysine (2.2 g/mg) as the most abundant amino acids. Thus, A. venetum flower could provide beneficial nutrient values for human health, e.g. adult weight maintenance or glucose homeostasis.