Generation of a new library of targeted mass spectrometry assays for accurate protein quantification in triple negative breast cancer (TNBC) tissues. Primary tumor tissue lysates from 105 TNBC patients treated at Masaryk Memorial Cancer Institute (MMCI) in Brno, Czech Republic, were used to generate the spectral library. This project covers raw files from data-dependent acquisition (DDA) – parallel accumulation-serial fragmentation (PASEF) measurements of 12 hydrophilic chromatography (HILIC) fractions of aliquot pool from complete set of 105 samples measured on timsTOF Pro; raw files of 16 individual samples measured in data-independent acquisition (DIA) – PASEF mode and used for hybrid library generation and for demonstrative quantitative DIA data extraction; Pulsar archive generated in Spectronaut 16.0 from 12 DDA-PASEF measurements of HILIC fractions and from 16 data-independent acquisition DIA-PASEF measurements of individual samples. The 16 DIA-PASEF runs of individual samples used for library generation were analyzed using newest versions of Spectronaut (version 18.5) and DIA-NN (version 1.8.1) software tools in library-based setting using the newly generated library as well as in library-free setting showing library-based method to outperform the use of predicted libraries in the terms of identification numbers.