Abstract: Microalgae are commercially important for the different industries as a natural source of a wide range of food and novel value‐added products. The adaptability of microalgae to produce different kind of pigments is gaining interest as a sustainable source of natural carotenoids. Astaxanthin is a popular ketocarotenoid with many health benefits. Microalgae is the potential source of astaxanthin. However, carotenoids production from microalgae in present time is reliant upon the requirement of strict culture conditions which are hard to maintain. However, the cultural conditions must be optimized first to increase strains’ productivity. In this context, Desmodesmus sp. PLM2 was isolated from the freshwater pond and screened for astaxanthin production under two-stage cultivation. The growth and carotenoid production were increased under optimized culture conditions compared to the control (standard BG-11 media). The optimized culture conditions were: 20 mM sodium nitrate; 3 mM glucose; 32 mM potassium chloride; pH 7, and temperature 27°C. In the second culture stage, the introduction of stress conditions induced the accumulation of orange-red carotenoid, later identified as astaxanthin using UHPLC. These results proved two-stage culture strategy as an effective way to optimise the carotenoid production in the newly isolated Desmodesmus sp. PLM2.