The endoplasmic reticulum (ER) is a network of membranes that regulates different cellular processes such as protein and lipid biogenesis, Ca2+ homeostasis and transport. Within neurons, the ER surface is huge and forms distinct structures depending on its cellular localization. Dense stacks of ER membranes, known as cisterna, have been identified in neurons, notably within the axonal initial segment (AIS, cisternal organelle) and dendritic spines (spine apparatus). While the specific functions of these cisternal structures are not fully understood, their role in spines is associated with calcium storage and synaptic plasticity. The cisternal organelle in the AIS, despite being recognized for some time, remains less explored regarding its contribution to axonal functionality. In this work, we are taking advantage of molecular tools to decipher the protein composition of the cisternal organelle. Due to the lack of a good marker, we optimized the split-APEX technology to perform mass spectrometry on a sample (rodent neuron cultures) enriched for cisternal organelles. Our investigation also includes the developmental dynamics of these structures, uncovering previously unreported patterns. Altogether, these data allows moving forward in discovering what is the role of the cisternal organelle in the AIS and ultimately in neurotransmission.