In humans, other mammals, and also in Drosophila, the paternal genome in the sperm is highly condensed and organized mainly in a protamine-based chromatin structure. However, the timing and mechanism of the switch from a histone- to the protamine-based chromatin configuration is still poorly understood. We therefore established Drosophila in vitro cultures of cysts with 64 synchronously developing spermatids genetically marked with histone H2AvD-RFP and ProtamineB-eGFP. Live cell imaging showed that the switch from H2AvD-RFP to Protamine-eGFP chromatin takes approximately five hours, with a short but clear overlap of the presence of both histones and protamines. Moreover, cultured pupal testes showed H4 hyperacetylation at the canoe stage shortly before histone removal; a feature previously observed in the intact animal. We then used TSA to inhibit histone deacetylation and found that premature hyperacetylation was already induced at the round nuclei stage of spermatids. However, this premature hyperacetylation did not lead to a premature switch to the protamine-based chromatin structure, showing that histone hyperacetylation is not the sole inducer of the histone to protamine switch. Importantly, we observed that inactivation of histone acetyltransferases by anacardic acid blocks further differentiation and thus prevents the degradation of histones and the switch to a protamine-based chromatin. Thus, we conclude that H4 hyperacetylation is an essential feature but not the sole inducer of the histone to protamine switch during spermiogenesis.