The aim of this study was to investigate the role of endothelial activation and glycocalyx degradation in the pathogenesis of feline panleukopenia (FPL) using biomarkers and to determine the diagnostic and prognostic significance of these biomarkers. Thirty cats with FPL and 10 healthy cats were enrolled. Clinical examination, blood gases, and complete blood count (CBC) were performed at enrollment. Endothelin-1 (ET-1), asymmetric dimethylarginine (ADMA), vascular endothelial growth factor-A (VEGF-A), and syndecan-1 (Syn-1) concentrations were measured using feline specific commercial enzyme-linked immunosorbent assay (ELISA) kits to assess endothelial glycocalyx damage. Nineteen (63.3%) of the cats with FPL recovered and survived, while 11 (36.7%) died. In cats with panleukopenia, acidemia was the most important blood gas finding, while leukopenia, lymphopenia, monocytopenia, granulocytopenia, and thrombocytopenia were the most dominant CBC findings. ET-1, ADMA, VEGF-A, and Syn-1 concentrations were significantly higher in cats with panleukopenia (p < 0.01). Serum Syn-1 and ET-1 concentrations were found to be useful in predicting mortality. In conclusion, the fact that the concentrations of all endothelial glycocalyx biomarkers (ET-1, ADMA, VEGF-A, Syn-1) were higher in cats with panleukopenia compared to healthy cats suggests that endothelial glycocalyx damage occurs during panleukopenia infection. In addition, Syn-1 and ET-1 were found to be potential prognostic factors with high sensitivity and specificity.