Microalgae are valuable sources of bioactive compounds. However, their production requires strategies to enhance metabolic responses. This study explores how Chlorella vulgaris responds to different salinity conditions using a two-stage cultivation strategy, assessing the change in amino acid and carotenoid content on microalgae over time. First, microalgae were cultivated under optimal conditions, followed by exposure to different salinity levels (150 mM and 300 mM NaCl). Growth kinetics, nutrient uptake, and biochemical composition were analysed, revealing distinct salinity-induced responses. Similar specific growth rates were achieved across all assays, while nitrate removal improved under salinity and phosphate uptake decreased. Amino acid profiling showed significant declines in the content of several compounds and carotenoid content also presented declining trends, although moderate salinity mitigated degradation in key pigments. Principal component analysis identified high correlations between amino acids and carotenoids contents, forming groups of compounds with similar variations. These findings contribute to a better understanding of the salinity-induced response of C. vulgaris, offering insights for biotechnology applications. By optimising cultivation conditions, salinity could enhance bioactive compound retention, supporting the development of sustainable microalgae-based products.