Withania somnifera L. (Solanaceae), for over 3000 years, has been considered an essential herb in Ayurvedic medicine. The roots of W. somnifera contain metabolites mainly belonging to steroidal lactones called withanolides, which possess various pharmacological activities such as neuroprotective, cardioprotective, anti-diabetic, antioxidant and anti-inflammatory. Since the demand on the market for W. somnifera extracts is increasing, with the aim to find an ecological and environmentally friendly strategy of extraction, the roots were submitted to different extraction techniques (macerations, ultrasound-assisted extraction and solid-liquid dynamic extraction) using EtOH:H2O 50:50, 75:25, 100:0. W. somnifera extracts were investigated by an integrated LC-ESI/QExactive/MS/MS and NMR approach to obtain comprehensive metabolite profiles. Principal Component Analysis of LC-MS and NMR data revealed how the extraction method and the solvent can affect the chemical profile of the extracts. Extracts obtained by maceration exhibited the highest amount of withanolides and withanosides, while the SLDE-Naviglio EtOH extract showed the highest amount of metabolites as benzoic acid, tropane alkaloids and sarcosine, reported for their CNS activity. Moreover, based on the use of this plant in the treatment of neurological disorders, the tyrosinase inhibitory activity of all the extracts was herein tested by spectrophotometric assay, showing IC50 values in a range of 32.86-85.36 µg/ml.