Traditional knowledge guides the use of plants for restricted therapeutic indications, but their pharmacological actions may be found beyond their ethnic therapeutic indications employing emerging computational tools. In this context, the present study was envisaged to explore the novel pharmacological effect of Achyranthes aspera (A. aspera) using PASS and PharmaExpert software tools. Based on the predicted mechanisms of the antidepressant effect for all analysed phytoconstituents of A. aspera, one may suggest its significant antidepressant action. The possible mechanism of this novel pharmacological effect is the enhancement of serotonin release, in particular caused by hexatriacontane. Therefore, pharmacological validation of the methanolic extract, hexatriacontane rich (HRF) and hexatriacontane lacking fraction (HLF) of A. aspera was carried out using the Forced Swimming Test and Tail suspension test in mice. The cortical and hippocampal monoamine and their metabolite levels were measured using high performance liquid chromatography (HPLC). A. aspera methanolic extract, HRF treatments showed a significant antidepressant effect comparable to imipramine. Further, the corresponding surge in cortical and hippocampal monoamine and their metabolite levels was also observed with these treatments. In conclusion, A. aspera has shown a significant antidepressant effect, possibly due to hexatriacontane, by raising monoamine levels.