The exploitation of plant by products is particularly important for the industries that produce bitter liqueurs, being the subsequent disposal very expensive. In fact, these wastes can be considered a good source of bioactive compounds that can be recycled due to favourable nutritional or biological properties. A new, sensitive and reliable HPLC-MS/MS method was described for the first time for the simultaneous determination of 18 bioactive compounds in the by-product of a bitter liqueur, which is particularly rich in gentiopicroside. Moreover, the waste extracts showed no cytotoxic activities against human fibroblasts (A549). The scavenging activity of ethanol:ethyl acetate (1:1) extract of the waste (S2, IC50=3.47μg/ml) was particularly high, together with its total phenolic content. Finally, this by-product extract showed synergistic effects with nerve growth factor (NGF) by inducing proliferation and differentiation of rat pheochromacytoma (PC12 Adh) cells. Results showed that by-products obtained during bitter liqueurs production represent a source of valuable bioactive compounds to be used in food, pharmaceutical and cosmetic industries.