Background: Molecular profiling of circulating tumor DNA (ctDNA) by NGS has demonstrated the value of liquid biopsy in informing treatment decisions and monitoring disease progression in cancer patients. Here we report results from the real-world clinical application of an ultrasensitive amplicon-based NGS liquid biopsy assay for advanced cancers. Methods: Plasma cell-free DNA (cfDNA) from 1,338 consecutive samples (51.3% lung, 15.6% breast, 8.4% colorectal, 26.8% from 18 other cancer types; 86.3% of cancer samples were metastatic) underwent real-world testing in a Singapore-based, CAP-accredited, CLIA-certified laboratory from Jan 2018 – Nov 2020. Genomic alterations were analyzed using an amplicon-based NGS assay that detects alterations in 80 cancer-related genes, microsatellite instability (MSI) and cancer-causing viruses, with previously validated cfDNA detection limits of 0.1% variant allele frequency (VAF) for SNPs and indels, 5% tumor fraction, and 2 IU/mL plasma, respectively. Results: ctDNA was successfully detected in 70.0% of cancer samples (76.3% from metastatic and 24.7% from localized tumors) for a total of 977 unique variants. Across ctDNA-positive samples the median VAF was 1.4%, and 8.5% of all reported variants had VAFs between 0.01 – 0.09%. The most frequently altered genes were TP53 (48.8%), EGFR (36.9%), KRAS (17.4%), PIK3CA (15.3%), and APC (9.5%). In ctDNA-positive lung cancer, 74.6% of samples (66.5% EGFR+) harbored ≥1 actionable target, with 29.9% of patients treated with a first- or second-generation EGFR tyrosine kinase inhibitor harboring an EGFR T790M resistance mutation. Among breast cancers, ESR1 or PIK3CA alterations predicting drug resistance/response were detected in 44.2% of HR+ cases. In colorectal cancer, alterations implicated in anti-EGFR resistance (NRAS, KRAS, BRAF, ERBB2) were found in 50.0% of samples. Profiling of cfDNA also enabled cancer monitoring, even among cancers with low genetic biomarker prevalence. 50% of nasopharyngeal cancer samples were Epstein-Barr virus-positive while 35.7% of liver cancer samples were Hepatitis B virus-positive, enabling an additional yield of 14.3% and 7.1% respectively, over ctDNA detection. cfDNA profiling of cancers of unknown primary origins informed potential tumor origins via the detection of tumor type-specific alterations such as mutations in AR, ESR1, GNAS,EGFR genes, viral biomarkers, and MSI. Conclusions: The use of a comprehensive pan-cancer liquid biopsy panel with rational inclusion of actionable and driver genes resulted in detection of ctDNA in the vast majority of clinical cases processed routinely. These findings support the utility of ultrasensitive amplicon-based NGS liquid biopsy assays in probing cancer drug sensitivity/resistance, tumor burden monitoring, and molecular characterization of tumors.