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Background Cannabis sativa is known to produce a class of terpenophenolic compounds named cannabinoids. The two main ones are cannabidiol (CBD) and tetrahydrocannabinol (THC), which have therapeutic properties. In the development of cannabis-based preparations, it is important to have suitable analytical methods for the analysis of the principal cannabinoids. Objective This study aimed to develop and validate a simple and rapid HPLC method with photodiode array detection for determination of CBD and THC in Cannabis sativa oil extract and infused ice cream, including a stability study. Method Chromatographic separation of CBD and THC was performed with a C18 column, with a mobile phase consisting of acetonitrile and water with formic acid (80 + 20 v/v) in isocratic elution mode, with detection at 208 nm for CBD and 280 nm for THC and 1.0 mL/min flow rate. Results The method was linear over a range of 1–5 µg/mL for CBD, and 20–100 µg/mL for THC; the relative standard deviation was <3.6%, the recovery ranged between 98.8 and 102.5% for oil and between 84 and 94% for ice cream, QL was 0.33 µg/mL for CBD and 2.30 µg/mL for THC, and the assay demonstrated adequate selectivity. CBD and THC were stable for at least 28 days under light protection at 22°C, 4°C, and -20°C in the oil and for at least 60 days at -20°C in the ice cream. Conclusions The results showed that the method was suitable for quantitative determination of CBD and THC in Cannabis sativa oil extract and infused ice cream, and it is useful for quality control purposes. Highlights The method is simple and fast, and it is useful for the quality control of a new product corresponding to an ice cream based on a Cannabis sativa oil extract. [ABSTRACT FROM AUTHOR]