In vitro culture has become a dependable approach for the mass production of plant material as the market for innovative plant-derived medicinal approaches has grown significantly. Furthermore, because it permits manipulation of biosynthetic routes to boost the production and accumulation of certain compounds, this technology has enormous potential for the manufacture of natural bioactive chemicals. As a result, the goal of this study was to develop an efficient micropropagation system for biomass production and to investigate the accumulation of bioactive compounds from Vaccinium corymbosum L., Duke and Hortblue Petite cultivars. Two in vitro plant tissue culture systems were used for shoots production: a solid medium (5 g/L Plant agar) and liquid medium (Plantform bioreactor). The culture medium used was Woddy Plant Medium (WPM) supplemented with two growth regulators: 0.5 mg/L and 1 mg/L zeatina (Z) and 5 mg/L N6-(2-Isopentenyl) adenine (2iP). The content of phenolic compounds, carotenoids, and chlorophylls of the in vitro shoot extracts were examined via the HPLC-DAD-MS/MS technique. The results showed that cv. Hortblue Petite produced a higher amount of biomass compared with cv. Duke, on all variants of culture media in both systems (solid and liquid), while the shoots extract of the Duke variety in the liquid culture system (under all concentrations of growth regulators) had the highest content of total phenolic compounds (16,665.61 ± 424.93 μg/g). In the case of the lipophilic compounds analysed (chlorophylls and carotenoids), the solid medium reported the highest values, whereas media supplemented with 0.5 mg/L Z was proved to have the richest total content for both cultivars.