ABSTRACT In vitro rearing protocols already established for honeybees are currently being adapted to assess the risk of pesticides, and to conduct comparative developmental biology studies on stingless bees. However, differences in critical life-history traits (development time and the type of larval nutrition leading to caste differentiation process) among social bees require the development of an in vitro rearing protocol for each species and caste. We generated a protocol to produce workers of Frieseomelitta varia (Lepeletier, 1836), a non-endangered and highly eusocial pollinator species with wide geographical distribution. We tested the viability of using either the eggs or the first instar larvae as the starting point for in-vitro transfer. In vitro rearing was performed in acrylic plates at 30 °C and 99% relative humidity during the larval feeding phase. The humidity was subsequently reduced to 75% during the following days of development. The experimental larvae were offered either 25 µL or 27 µL of larval food. The development time, emergence and mortality rates, and morphological parameters of the emerged workers were assessed. In the process of validating the protocol, the adults that emerged after in vitro rearing were compared with colony-reared adults. In our results, 27 µL of larval food allowed 90% of workers to emerge. No significant differences were found between the emerging workers reared in vitro and those reared in the colony. The described protocol is a useful method for rearing F. varia workers in vitro, which can be used for diverse types of experimental approaches.