Background: Since different culturing parameters – such as media composition or cell density – lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons – both in vivo and in vitro – is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes. New method: Our culturing protocol is based on a novel serum-free preparation of astrocyte – conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7,14 and 60 days in vitro (DIV). Results: ACM-based cultures gave the best results for all tested criteria, i.e. growth cone’s size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. Comparison with existing method(s): ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. Conclusions: We propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.