Nutrition models have improved in terms of predicting AA requirements and their supply for diet formulation. However, data have demonstrated the essential AA (EAA) content of feeds or animal products are not adequately described using 24h hydrolysis time. Thus, the objective was to evaluate the EAA content of milk, tissues, and feeds after multiple hydrolysis times, from 2 h to 168 h, and determine a correction factor for each AA. Twenty-six feeds were chosen, as well as 6 tissue samples representing whole body composition of Holstein heifers from serial harvest studies. The milk sample was pooled from 4 bulk tank samples over 3 d from the Cornell University Dairy. Feed samples were analyzed for all EAA by HPLC following acid hydrolysis at 110°C in a block heater for 2, 4, 6, 12, 18, 21, 24, 30, 48, 72, 120 and 168h. Performic acid pre-oxidation was conducted for the sulfur AA and barium hydroxide was used for Trp hydrolysis. Tissues and milk were analyzed for 21, 72 and 168h using the same methods. A least-squares nonlinear regression was used to determine the true AA content of the samples and significance declared at P < 0.05. The EAA of feeds, milk, and tissues continued to increase after 24h and overall, had the greatest EAA concentration after 168h hydrolysis (P < 0.05). The branched chain AA (BCAA) for most feeds, and all milk and tissue samples increased from 24h to 168h hydrolysis (P < 0.05). Lys increased in concentration up to 168h for most feeds (P < 0.05) but decreased in milk after 72 h and tissue after 21 h (P < 0.002). Two ratios, A₀/24h and Max/24h, were determined and compared among sources to be used as correction factors for the 24h hydrolysis. The A₀ is the AA concentration at 0h of hydrolysis and Max being the maximal concentration of AA. The ratios for BCAA vary among sources of EAA and different ratios should be determined for each source to have an accurate correction factor. In summary, a single time point hydrolysis is not an accurate representation of the EAA concentration of a substrate and correction factors could be used to determine the actual EAA content by substrate.