The aims of this prospective study were to describe the normal sonographic size and appearance of the feline spleen, and to test effects of sevoflurane anesthesia and blood collection on sonographic characteristics. Sonographic evaluations were performed in 60 healthy blood donor cats prior to anesthesia and blood collection (baseline), after anesthesia induction, after completion of blood collection while under anesthesia, and 7–10 days after blood collection. Transverse sonographic images of the splenic mid-body were made. Splenic height, width, cross-sectional area, echogenicity relative to the left renal cortex, and echotexture were recorded. Height, width, and cross-sectional area were compared across time periods using linear-mixed effects models. Holm's method was applied to adjust for multiplicity and control the overall Type 1 error rate at α = 0.05. Normal transverse sonographic splenic measurements (mean ± standard deviation) at baseline were: height 8.2 ± 1.4 mm; width 26.7 ± 4.4 mm; and CSA 1.6 ± 0.5 cm2. While there were statistically significant differences in splenic height, width, and cross-sectional area after sevoflurane anesthesia and/or blood collection, differences were of low magnitude. The spleen was diffusely homogenous and subjectively isoechoic or mildly hyperechoic relative to the left renal cortex. In 77% of castrated male cats, the spleen was isoechoic to the left renal cortex. We conclude that sevoflurane anesthesia and blood donation do not subjectively alter splenic dimensions, echogenicity and echotexture in normal cats.