Background: In metastatic PC, tracking genomic alterations over time and with treatment can predict disease outcomes, guide treatment, and identify novel therapeutic targets. Analysis of peripheral blood ctDNA offers a non-invasive liquid biopsy for genomic profiling, but ctDNA often comprises a small portion of total cell-free DNA (cfDNA) in the blood, requiring high depth of sequencing which may still miss PC-relevant variants. We and others have shown that single CTCs captured from the same blood sample yield additional PC-relevant genomic data when combined with ctDNA profiling, but currently there is no assay that can be applied uniformly to both analytes. Here, we present a new amplicon-based sequencing panel capable of detecting PC-relevant variants in both cfDNA and single CTCs. Methods: PC-relevant genes were curated by overlapping reviews of cBioPortal for cancer genomics, the catalogue of somatic mutations in cancer (COSMIC), and PC-relevant genomic literature. HERCULES targets 36 genes with 258 amplicons covering PC-associated mutations and copy number variations. AmpliSeq HD targeted sequencing technology was applied for analysis of both cfDNA and CTCs. Analytical validation was performed sequentially, first using AcroMetrix and Seracare DNA control samples, then single and pooled LNCap and PC3 PC cells and cfDNA, then 8 patient peripheral blood samples of matched plasma cfDNA and single CTCs captured using the RareCyte platform from participants in SWOG S1802, an ongoing CTEP/CIRB-approved phase 3 trial for men with metastatic PC. Results: In DNA control samples with allele frequencies of 1%, 0.25%, and 0.1%, HERCULES displayed sensitivity of 100%, 75%, and 50%, respectively. In matched single cells and cfDNA from cell lines, HERCULES detected 100% of database-annotated somatic variants in cfDNA and in pools of 2 or more cells, and it identified >90% of variants in single cells. In 16 matched patient samples of cfDNA and single CTCs, with read coverage depths of >50,000x for 20ng cfDNA and >1,500x for single CTCs, HERCULES detected shared variants (mean N per sample = 6.1) as well as unique somatic variants (mean of 1.1 CTC variants not called in the cfDNA, and 4.1 cfDNA variants not called in the CTCs). Conclusions: HERCULES is, to our knowledge, the first PC-specific targeted sequencing panel capable of parallel genomic profiling of both ctDNA and CTCs. In our preliminary analytical validation, it detected somatic variants with high sensitivity in matched cfDNA and CTC samples. With additional validation, HERCULES has the potential to augment conventional ctDNA profiling by illuminating both cell-free and cellular genomic evolution during PC treatment and progression.