A widely used method for protein identification couples prefractionation of protein samples by one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) with liquid chromatography-tandem mass spectrometry (LC/MS/MS). We developed a new label-free quantitative algorithm by combining measurements of spectral counting, ion intensity and peak area on 1D-PAGE based proteomics. This algorithm has several improvements over other label-free quantitative algorithms: (i) Errors in peak detection are reduced since the retention time is based on each LC/MS/MS run and actual precursor m/z. (ii) Detection sensitivity is increased since protein quantification is based on the combination of peptide count, ion intensity, and peak area. (iii) Peak intensity and peak area are calculated in each LC/MS/MS run for all slices from 1D-PAGE for every single identified protein and visualized as Western blot image. The sensitivity and accuracy of this algorithm were demonstrated by using standard curves (17.4 fmol - 8.7 pmol), complex protein mixtures (30 fmol - 1.16 pmol) of known composition, and spiked protein (34.8 fmol - 17.4 pmol) in complex proteins. We studied the feasibility of this approach using the secretome of angiotensin II (Ang II) stimulated vascular smooth muscle cells (VSMC). From the VSMC conditioned medium, 629 proteins were identified including 212 putative secreted proteins. Twenty-six proteins were differently expressed in control and Ang II-stimulated VSMC, including 18 proteins not previously reported. Proteins related to cell growth (CYR61, NOV, and clusterin) are increased while growth arrest specific 6 (GAS6) and growth/differentiation factor 6 are decreased by Ang II stimulation. Ang II-stimulated changes of plasminogen activator inhibitor-1, GAS6, cathepsin B, and periostin were validated by Western blot. In conclusion, a novel label-free quantitative analysis of 1D-PAGE-LC/MS/MS based proteomics has been successfully applied to the identification of new potential mediators of Ang II action and may provide an alternative to traditional protein staining methods.