Obesity is a chronic disease that negatively affects life expectancy through its association with life-threatening diseases such as cancer and cardiovascular diseases. Expression proteomics combined with in silico interaction studies are used to uncover potential biomarkers and the pathways that promote obesity-related complications. These biomarkers can either aid in the development of personalized therapies or identify individuals at risk of developing obesity-related diseases. To determine the serum protein changes, Wistar rats were fed standard chow (low fat, LF), or chow formulated high fat (HF) diets (HF1, HF2 and HF3) for 8 and 42 weeks to induce obesity. Serum samples were collected from lean and obese rats at these time points. The serum samples were precipitated using trichloroacetic acid (TCA)/acetone and analyzed by 2-Dimensional SDS-PAGE. Serum protein profiles were examined using mass spectrometry (MS)-based proteomics and validated by western blotting. Protein-protein interactions among the selected proteins were studied in silico using bioinformatics tools. Several proteins showed differences in expression among the three HF diets when compared to the LF diet, and only proteins with ≥ twofold expression levels were considered differentially expressed. Apolipoprotein-AIV (APOA4), C-reactive protein (CRP), and alpha 2-HS glycoprotein (AHSG) showed differential expression at both 8 and 42 weeks, whereas alpha 1 macroglobulin (AMBP) was differentially expressed only at 8 weeks. Network analysis revealed some interactions among the proteins, an indication that these proteins might interactively play a crucial role in development of obesity-induced diseases. These data show the variation in the expression of serum proteins during acute and chronic exposure to high fat diet. Based on the expression and the in-silico interaction these proteins warrant further investigation for their role in obesity development.