Risk assessment of pesticides involves ecotoxicological testing. In case pesticide exposure to bees is likely, toxicity tests are performed with honey bees (Apis mellifera), with a tiered approach, for which validated and internationally accepted test protocols exist. However, concerns have grown regarding the protection of non-Apis bees bumble bees (Bombus spp.), solitary and stingless bees, given their different life cycles and therefore distinct exposure routes. Larvae of solitary bees of the genus Osmia feed on unprocessed pollen during development, yet no toxicity test protocol is internationally accepted or validated to assess the impact of pesticide exposure during this stage of their life cycle. Therefore, the purpose of this study is to further validate a test protocol with two solitary bee species (O. cornuta and O. bicornis) to assess lethal and sublethal effects of pesticide exposure on larval development. Larvae were exposed to thiacloprid (neonicotinoid insecticide) mixed in a new, artificial pollen provision. Both lethal (developmental and winter mortality) and sublethal endpoints (larval development time, pollen provision consumption, cocoon weight, emergence time and adult longevity) were recorded. Effects of lower, more environmentally realistic doses were only reflected in sublethal endpoints. In both bee species, thiacloprid treatment was associated with increased developmental mortality and larval development time, and decreased pollen provision consumption and cocoon weight. The test protocol proved valid and robust and showed that for higher doses of thiacloprid the acute endpoint (larval mortality) is sufficient. In addition, new insights needed to develop a standardized test protocol were acquired, such as testing of a positive control for the first time and selection of male and female individuals at egg level. Display omitted •Including solitary bees in pesticide risk assessment is proven to be possible.•Both lethal and relevant sublethal effects can be assessed simultaneously.•Dose-response relationships due to the insecticide thiacloprid have been shown.•Chronic thiacloprid uptake of Osmia spp. larvae is reflected in sublethal endpoints.•Larval development time increases with increasing thiacloprid exposure.