Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process whose loss-of-function has been linked to a growing list of pathologies. Knockout mouse models of key autophagy genes have been instrumental in the demonstration of the critical functions of autophagy, but they display early lethality, neurotoxicity and unwanted autophagy-independent phenotypes, limiting their applications for in vivo studies. To avoid problems encountered with autophagy-null transgenic mice, we investigated the possibility of disturbing autophagy pharmacologically in the long term. Hydroxychloroquine (HCQ) ip injections were done in juvenile and adult C57bl/6j mice, at range doses adapted from the human malaria prophylactic treatment. The impact on autophagy was assessed by western-blotting, and juvenile neurodevelopment and adult behaviours were evaluated for four months. Quite surprisingly, our results showed that HCQ treatment in conditions used in this study neither impacted autophagy in the long term in several tissues and organs nor altered neurodevelopment, adult behaviour and motor capabilities. Therefore, we recommend for future long-term in vivo studies of autophagy, to use genetic mouse models allowing conditional inhibition of selected Atg genes in appropriate lineage cells instead of HCQ treatment, until it could be successfully revisited using higher HCQ doses and/or frequencies with acceptable toxicity.