The objective of this study was to investigate in-vitro exposure of healthy feline red blood cells to venom of four species of rattlesnakes and to evaluate the presence and type of echinocytes that form. Blood from 10 healthy felines was collected and within 30 min exposed to four species of rattlesnake venom. Cytologic evaluation for echinocytes was performed at 0-, 15-, and 30-min post-exposure to each of the species' venom. There was significant formation of echinocytes in a time dependent manner with in-vitro exposure of red blood cells to rattlesnake venom. Cytologic evaluation of blood smears at 15 and 30 min showed a progressive increase in echinocyte numbers over time, with a significantly higher number of echinocytes formed at both timepoints compared to the 0-min timepoint. Furthermore, the morphology of the echinocytes differed based on the length of time exposed to the venom, with type I echinocytes being significantly present early on post-exposure (0 and 15 min) and type III echinocytes being most prevalent later post-exposure (15 and 30 min). There was not a significant difference in the formation of echinocytes between the rattlesnake species. This study demonstrates that there is a time dependent formation of echinocytes in feline blood post-exposure to four species of rattlesnake venom in the in-vitro setting. Cytologic evaluation of blood smears in cats with suspected rattlesnake envenomation to evaluate for echinocyte populations may use to provide support for the diagnosis and to direct the timely administration of appropriate treatments.