In this paper, we present a fully automated tool, called IDEAL-Q, for label-free quantitation analysis. It accepts raw data in the standard mzXML format as well as search results from major search engines, including Mascot, SEQUEST, and X!Tandem, as input data. To quantify as many identified peptides as possible, IDEAL-Q uses an efficient algorithm to predict the elution time of a peptide unidentified in a specific LC-MS/MS run, but identified in other runs. Then, the predicted elution time is used to detect peak clusters of the assigned peptide. Detected peptide peaks are processed by statistical and computational methods and further validated by Signal-to-noise ratio, Charge state, and Isotopic distribution criteria (SCI validation) to filter out noisy data. IDEAL-Q’s performance has been evaluated by several experiments. First, a serially diluted protein mixed with E. coli lysate showed a high correlation with expected ratios and demonstrated good linearity (R2=0.996). In a biological replicate experiment on the THP-1 cell lysate, IDEAL-Q quantified 87% (1,672 peptides) of all identified peptides, surpassing the 45.7% (909 peptides) achieved by the conventional identity-based approach, which only quantifies peptides identified in all LC-MS/MS runs. Manual validation on all 11,940 peptide ions in 6 replicate LC-MS/MS runs revealed that 97.8% of the peptide ions were correctly aligned and 93.3% were correctly validated by SCI. Thus, the mean of the protein ratio, 1.00±0.05, demonstrates IDEAL-Q’s high accuracy without human intervention. Finally, IDEAL-Q was applied again to the biological replicate experiment with an additional SDS-PAGE fractionation step, to show its compatibility for label-free experiments with fractionation. For flexible workflow design, IDEAL-Q supports different fractionation strategies and various normalization schemes, including multiple spiked internal standards. User-friendly interfaces are provided to facilitate convenient inspection, validation, and modification of quantitation results. In summary, IDEAL-Q is an efficient, user-friendly, and robust quantitation tool.