This investigation was conducted to develop a protocol for shoot proliferation in Khaya senegalensis (Desr.) A. Juss., as a multipurpose tree with commercial and environmental values. MS culture medium with or without 2gl-1 activated charcoal (AC) and benzylladenine (BA) (0.0, 0.5 and 1.0 mgl-1) individually or combined with indol-3-butyric (IBA) (0.0, 0.05, 0.1 and 0.2 mgl-1) was used. Polyethylene glycol (PEG) was added at various levels (0.0, 0.5, 1.0, 1.5, and 2.0%) to the optimization culture medium and melatonin at 0, 1, and 2 mgl-1. Using MS medium supplemented with 1.0 mgl-1 of BA + 0.05 mgl-1 of IBA resulted in the most proliferated shoots with the highest rooting percentage at the presence of 2 gl-1 of AC. The enhancement effect of melatonin at 2 mg/l was noticed on proliferated shoots under 0.5 and 1% of PEG. Melatonin treatments resulted in a significant increase in photosynthetic pigment contents (P≤0.05). The highest concentrations of PEG (2%) and melatonin (2 mgl-1) individually or interacted were more effective for increasing secondary metabolites such as total phenol, tannin, flavonoid, and antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD). This study could be useful in mitigating drought stress using melatonin as a regulator for physiological and biochemical processes.