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Protein hydrolysates are an important part of the human diet. Often, they are prepared from milk, soy, or collagen. In the present study, four different collagen hydrolysates were tested, varying in the average molecular weight and the animal source. Three types of samples, the dissolved start products, in vitro generated dialysates (containing the digested components that are potentially available for small intestinal absorption), and human serum collected after product ingestion, were analyzed using LC-MS to compare the state of the hydrolysates before and after absorption, i.e., uptake into the blood. It was found that the composition of the collagen hydrolysates prior to and after ingestion was highly complex and dynamic, which made it challenging to predefine a strategy for a targeted analysis. Therefore, we implemented a new analytical approach to first map hydrolysate data sets by performing non-targeted LC-MS analysis followed by non-targeted and targeted data analysis. It was shown that the insight gained by following such a top down (data) analytical workflow could be crucial for defining a suitable targeted setup and considering data trends beyond the defined targets. After having defined and performed a limited targeted analysis, it was found that, in our experimental setup, Hyp-Gly and especially Pro-Hyp contributed significantly as carrier to the total Hyp increase in blood after ingestion of collagen hydrolysate. [ABSTRACT FROM AUTHOR]