Opioid and local anaesthetic receptors are abundantly concentrated in different layers of the skin. Therefore, simultaneous targeting of these receptors can produce more potent dermal anaesthesia. Herein, we developed lipid-based nanovesicles for the co-delivery of buprenorphine and bupivacaine to efficiently target skin-concentrated pain receptors. Invasomes incorporating two drugs were prepared by ethanol injection method. Subsequently, the size, zeta potential, encapsulation efficiency, morphology, and in-vitro drug release of vesicles were characterized. Ex-vivo penetration features of vesicles were then investigated by the franz diffusion cell on the full-thickness human skin. Wherein, it was demonstrated that invasomes penetrated the skin deeper and delivered bupivacaine more effectively than buprenorphine to the target site. The superiority of invasome penetration was further evidenced by the results of ex-vivo fluorescent dye tracking. Estimation of in-vivo pain responses by the tail-flick test revealed that compared with the liposomal group, the group receiving invasomal formulation and drug-free invasomal formulation (only containing menthol) displayed increased analgesia in the initial times of 5 and 10 min. Also, no signs of oedema or erythema were observed in the Daze test in any of the rats receiving the invasome formulation. Finally, ex-vivo and in-vivo assays demonstrated efficiency in delivering both drugs into deeper layers of skin and exposing them to the located pain receptors, which improves the time of onset and the analgesic effects. Hence, this formulation appears to be a promising candidate for tremendous development in the clinical setting.