Abstract: With the rapid development of mass spectrometry technology, it has been widely used in proteomic analysis of trace protein samples. To date, the effectiveness of filter-aided sample preparation (FASP) and in-stage tip (iST) protocols in microprotein sample repreparation has not been thoroughly investigated. Although a number of researches have been performed to compare the capacity of the two enzyme digestion protocols for identifying the proteins, no studies have been conducted when the protein input is lower than 1 μg, and the depth of analysis is shallow in terms of subsequent data analysis. In the present work, the optimal protein inputs of the two enzyme digestion schemes for 0.2-10 μg microprotein samples were studied, and the FASP and iST enzyme digestion protocols under the same protein input amount were compared. This study also systematically evaluated the two enzyme digestion protocols in terms of the numbers of identified proteins and peptides, the coverage of amino acid sequences, the frequency of identification, the coefficient of variation (CV), and the linear correlation within the groups of experiment. For iST, when the protein input was 5 μg, the number of identified proteins reached the highest value. For FASP, the number of identified proteins increased with increase in the amount of protein input, while the magnitude of the increase decreased after the protein input reaches to 5 μg. Further, the stability of each protocol of each group was evaluated from three aspects, including the number of identified proteins with amino acid sequence coverage of no less than 20%, 3 times in the triplicate samples, and a coefficient of variation of less than 5%. When the protein input was 5 μg, the number of proteins identified by iST in the three stability assays was maximum, meaning the stability reached to the best. When the protein input increased from 0.2 to 10 μg, the number of proteins identified in the three stability assays of FASP gradually increased with increase in protein inputs, while the magnitude of the increase decreased when the protein input was larger than 5 μg, and the stability of the protocol tended to the best. Under the same protein input, when the protein input amount was no more than 1 μg, the iST digestion protocol was better than FASP in terms of the number of proteins identified and the stability of the analysis procedure. When the protein input was more than 1 μg, the result was opposite. In summary, by comparing the numbers of proteins identified by iST and FASP with the protein input ranging from 0.2 to 10 μg, the FASP digestion protocol leads to identify more proteins with better stability, and gene ontology (GO) analysis can provide richer biological information.