The isolation of bovine lipoproteins often involves ultracentrifugation and gel filtration chromatography; however, large sample volumes, lengthy run times, and high centrifugal forces and salt concentrations that may cause dissociation of lipoproteins are often applied. Alternatively, the utilization of size exclusion chromatography by fast protein liquid chromatography (FPLC) avoids these method inputs. Our objective was to utilize FPLC to characterize the lipid and protein composition of bovine lipoproteins during the periparturient period. Blood samples were collected from 25 peripartal Holstein dairy cows before feeding at d −12, 0, and 10, relative to parturition. Serum lipoproteins were isolated using FPLC and a size exclusion column. Measurement of total triacylglycerol (TAG), phospholipid (PL), cholesterol, and protein was performed using colorimetry. Data were analyzed as repeated measures using a mixed model (fixed effect of fraction and random effect of cow). Our approach revealed 4 distinct fractions: TAG-rich (VLDL), low-density (LDL), and large (buoyant) and small (dense) high-density lipoprotein (HDL) subclasses. VLDL primarily contained TAG (56, 31, and 35% of total components at d −12, 0, and 10, respectively). VLDL TAG levels were greater at d −12 (P < 0.01). LDL primarily contained PL (54, 52, and 55% of total components at d −12, 0, and 10, respectively) and cholesterol (42, 45, and 42% of total components at d −12, 0, and 10, respectively). LDL PL levels were lowest at parturition (P < 0.01). LDL cholesterol followed a similar pattern (P < 0.01). Buoyant HDL contained equal levels of PL and cholesterol (36 and 45% of total components, respectively), and buoyant HDL PL and cholesterol levels were lowest at parturition (P < 0.01). Protein levels were greatest in dense HDL (73% of total components; P < 0.01). Protein levels within buoyant HDL were greatest at d 10 (P < 0.05), whereas dense HDL protein levels were not modified by time. Because the observed lipoprotein composition is in agreement with previous work using alternative methods, we conclude that the use of FPLC is a means to isolate bovine lipoproteins from peripartal cows.