This research study was carried out in the plant tissue culture laboratory of the Agricultural Botany Department, Faculty of Agriculture, Ain Shams University, Shoubra El-khaima, Cairo, Egypt. Experiments were executed for the duration of two consecutive years 2017 and 2018 on chicory plant. Chicory (Cichorium intybus L.), which belongs to Asteraceae family, is considered as an important medicinal plant due to the presence of many bioactive substances such flavonoids, phenolic compounds, alkaloids, steroids, terpenoids, including( coumarines, cichoriin, esculetin, inulin, sesquiterpene lactones, chicoric acid, caffeic acid and some vitamins). In this research in vitro experiments were carried out using full strength Murashige and Skoog basal medium (MS) supplemented with different combinations of two plant growth regulators; Indole-3-butyric acid (IBA) including two concentrations (0.5 – 2.0 mg/l) and Naphthalene acetic acid (NAA) comprising four concentrations (0.5 – 2.0 – 3.0 – 5.0 mg/l). An abaxially (lower side) leaf explants (square pieces 0.5 × 0.5 cm) which were taken from 20 days old aseptic chicory seedlings were inoculated to (MS) surface. Initially, chicory seeds were aseptically germinated on half-strength MS medium, after surface sterilization by 70 % (v/v) ethanol for 60 seconds then soaking in 10 % Clorox (0.5% sodium hypochlorite NaOCl) for 10 min to produce the aseptic chicory seedlings which were the source of true leaf explants used in this research study. Total phenolic compounds and flavonoids content were extracted from six-week C. intybus friable callus produced under both light and dark in vitro culture conditions inside a growth chamber incubation room where temperature was adjusted at 25oC ±1. Total phenolic compounds and flavonoids were determined by spectrophotometric methods. The highest values for their contents were from chicory calli when MS callus induction medium was supplemented with 2 mg/l NAA under total dark condition when compared with the other remaining growth regulator treatment combinations and alternative light regime conditions.