Malaria parasites have complicated lifecycles involving both human and mosquito hosts. In ~8 days, a single parasite can form an oocyst in the mosquito midgut and produce thousands of daughter sporozoites. These sporozoites then egress from the oocyst and undergo a complex series of translocation and invasion events to reach the mosquito salivary gland. To facilitate host cell invasion, sporozoites use specialised secretory organelles known as rhoptries. The small size of these parasites and their rhoptries, however, has made it challenging to interrogate sporozoite rhoptry biology. To overcome this, we developed mosquito-tissue ultrastructure expansion microscopy (MoTissU-ExM), a technique that physically expands parasites and their hosts ~4.5-fold. Using MoTissU-ExM, we developed a timeline for sporozoite rhoptry biogenesis in the mosquito midgut and observed the changes they undergo during salivary gland invasion. We leverage these findings to characterise the role of rhoptry neck protein (RON) 11 in sporozoites, showing that RON11 disrupted sporozoites only have half the required number of rhoptries and that therefore RON11 is the only protein known to be involved in sporozoite rhoptry biogenesis. Further, we show that while these RON11-disrupted sporozoites can anatomically enter the salivary gland, they fail to invade salivary gland epithelial cells or the secretory cavity.