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The effect of increasing the trypsin amount in bottom-up based targeted protein analysis is evaluated. By applying an increased trypsin-to-protein ratio (1:1 (w/w)) after heat denaturation (60 °C), reduction and alkylation, the digestion time could be reduced profoundly compared to conventional digestion conditions (ratio 1:40, overnight) without compromising method sensitivity or digestion repeatability. The procedure was obtained after a systematic evaluation of trypsin level and trypsin quality using a set of three model proteins: human chorionic gonadotropin (hCG), bovine serum albumin (BSA) and cytochrome C (CytC). All peptides monitored were produced at similar or higher levels after 45 min at trypsin-to-protein ratio 1:1, compared to conventional overnight digestion (exception: CytC using modified trypsin, required up to 4 h (at 1:1 ratio) in order achieve this). Peptide decay due to chymotryptic activity was observed at longer digestion times, but the effect was circumvented using digestion times <4 h. The accelerated digestion protocol (1:1 (w/w), 45 min) was applied to realistic human serum samples containing the biomarker protein hCG to demonstrate its applicability. [ABSTRACT FROM AUTHOR]