Khoa is a milk concentrate made by simmering milk, primarily buffalo milk, until it thickens, and contains about 60-70% milk solids. In India’s tropical climate, milk spoils quickly, so around 50-55% of milk production is used to make traditional dairy sweets, with khoa being a key ingredient. Surplus or leftover milk is often used for this purpose. Although cow milk makes up about 30% of total milk production, it is less commonly used for khoa due to its lower solid-not-fat (SNF) and fat content. Due to limited research on cow milk khoa, a study was conducted to gather data on how the acidity development and neutralization of milk affect the quality of cow milk concentrate (khoa) made from fresh, sour, and neutralized cow milk. The study examined physical, compositional, and textural properties, SDS-PAGE, as well as the fatty acid profile during storage at 30°C for 7 days and 5°C for 21 days. The findings revealed that the developed acidity and subsequent neutralization of milk caused significant changes (p<0.05) in pH, ash, color, tyrosine value, lactulose, HMF, furosine, induction period, FFA content, peroxide value, and textural and sensory attributes. Over the storage period, acidity, ash content, tyrosine value, furosine, HMF, free fatty acids, peroxide value, TBA value, butyric acid, and stearic acid increased, while pH and oleic acid decreased. From a textural perspective, khoa made from neutralized milk was stickier and less hard compared to the control. Textural attributes significantly differed (p<0.05) in terms of lightness, redness, springiness, chewiness, and hardness values during storage. However, the fatty acid profile (except butyric acid), TBA value, SDS-PAGE analysis of caseins, and mineral composition (except sodium content) of altered khoa samples showed minimal nonconformity (p>0.05).