Phenols, important secondary metabolites in plants, are responsible for specific defence mechanisms against abiotic stress, due to their strong antioxidant activity. Flavonoids, as part of phenolic group, are also involved in plant stress responses, being primarily responsible for photo-protection against UV solar radiation. Based on these premises, the plant response to optimized in vitro culture was evaluated, by quantifying the total polyphenolic content, the total flavonoid content and the antioxidant activity, both under in vitro and ex vitro conditions. Four closely related potato genotypes were analysed: the wild species Solanum bulbocatanum, S. tuberosum cv. ‘Rasant’ and two somatic hybrids between them. For all genotypes, both total polyphenolic content and antioxidant activity were increasing under optimized in vitro culture. The shoot responses were genotype dependent and the two somatic hybrids were intermediate between the parents both as morphology and reaction to in vitro stress. The somatic hybrid 1508/5, having morphology similar to potato, was reacting as the wild species, while the somatic hybrid 1508/2, with a similar morphology to the wild species was reacting as potato to in vitro stress. The somatic hybrid 1508/5 being also resistant to late blight is of interest for further use in pre-breeding. Total flavonoid content is decreasing under in vitro as compared to ex vitro conditions, UV-B radiation, the major trigger of flavonoid biosynthesis being absent in the fluorescent light. This study reveals the effect of in vitro culture on flavonoid content and details aspects of the biochemical parameters involved in plant in vitro stress.