In vitro seeds germination and plantlets establishment of Cladanthus mixtus (L.) Oberpr. and Vogt were studied in this report. A reliable protocol was developed for in vitro seed germination, multiplication and plantlet regeneration of Cladanthus mixtus. The seeds were sterilized and cultured in different media. Among the two basal media evaluated for symbiotic seed germination, gelled distilled water was found to be the best with a high percentage of seeds germination (100%) after 33 hours of culture. Seedlings were further transferred to different types of media. Result observed after 4 weeks showed that MS medium promotes the highest growth with an average of 2.75 ± 0.12 cm shoot length and 2.60 ± 0.29 shoots per explants, and the mean number of roots achieved 3.33 ± 0.17 root per explants with a length of 2.42 ± 0.16 cm. This study showed that macroelements of MS (1962) medium is essential for in vitro shoot multiplication, growth and rooting of shoots of Cladanthus mixtus L.