Background: Cancer research often focuses on protein quantification in model cancer cell lines and cancer tissues. SWATH (sequential windowed acquisition of all theoretical fragment ion spectra), the state of the art method, enables the quantification of all proteins included in spectral library. Spectral library contains fragmentation patterns of each detectable protein in a sample. Thorough spectral library preparation will improve quantitation of low abundant proteins which usually play an important role in cancer. Aim: Our research is focused on the optimization of spectral library preparation aimed at maximizing the number of identified proteins in MCF-7 breast cancer cell line. First, we optimized the sample preparation prior entering the mass spectrometer. We examined the effects of lysis buffer composition, peptide dissolution protocol and the material of sample vial on the number of proteins identified in spectral library. Next, we optimized mass spectrometry (MS) method for spectral library data acquisition. Conclusion: Our thorough optimized protocol for spectral library building enabled the identification of 1,653 proteins (FDR < 1%) in 1 µg of MCF-7 lysate. This work contributed to the enhancement of protein coverage in SWATH digital biobanks which enable quantification of arbitrary protein from physically unavailable samples. In future, high quality spectral libraries could play a key role in preparing of patient proteome digital fingerprints.