Shoot tips of pineapple (Ananas Comosus) cultivar "Queen" were cultured individually on MS medium supplemented with 2.0 mg/l BA+0.1 mg/l NAA +0.4 mg/l Thiamine HCL+30 g/l sucrose +6 g/l agar for establishment stage. Sucrose at different concentration (30, 20 and 10 g/l) and stevia extract at different concentration (20, 13 and 7 ml/l) were tested on multiplication and rooting stage of pineapple in vitro plants. Moreover, different biochemical analysis such as total carbohydrate, total sugar, starch, phenol, flavonid, chlorophyll A, B and AB were estimated. The obtained results assured that sucrose at 30 g/l is an important source of carbon for shoot multiplication of pineapple. Meanwhile, during rooting stage, the highest roots number and roots length were obtained when using MS medium supplemented with 20 g/l sucrose + 7 ml/l stevia. This combination also increased total sugar, starch and chlorophyll B content of pineapple leaves. However, sucrose alone showed superiority in some chemical content under study (total carbohydrate, flavonoids and chlorophyll A). From this investigation we can conclude that sucrose is an important source of carbon for in vitro plants. However, stevia extract enhanced plantlets growth in rooting stage.