MOTIVATION: Post-acquisition sample normalization is a critical step in comparative metabolomics to remove the variation introduced by sample amount or concentration difference. Previously reported approaches are either specific to one sample type or built on strong assumptions on data structure, which are limited to certain levels. This encouraged us to develop MAFFIN, an accurate and robust post-acquisition sample normalization workflow that works universally for metabolomics data collected on mass spectrometry (MS) platforms. RESULTS: MAFFIN calculates normalization factors using maximal density fold change (MDFC) computed by a kernel density-based approach. Using both simulated data and 20 metabolomics datasets, we showcased that MDFC outperforms four commonly used normalization methods in terms of reducing the intragroup variation among samples. Two essential steps, overlooked in conventional methods, were also examined and incorporated into MAFFIN. (i) MAFFIN uses multiple orthogonal criteria to select high-quality features for normalization factor calculation, which minimizes the bias caused by abiotic features or metabolites with poor quantitative performance. (ii) MAFFIN corrects the MS signal intensities of high-quality features using serial quality control samples, which guarantees the accuracy of fold change calculations. MAFFIN was applied to a human saliva metabolomics study and led to better data separation in principal component analysis and more confirmed significantly altered metabolites. AVAILABILITY AND IMPLEMENTATION: The MAFFIN algorithm was implemented in an R package named MAFFIN. Package installation, user instruction and demo data are available at https://github.com/HuanLab/MAFFIN. Other data in this work are available on request. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.