Next-generation sequencing (NGS)-based comprehensive tumor profiling from liquid biopsy samples can significantly improve diagnosis and monitoring of tumors when high-quality tissue material is difficult to obtain. In addition, it offers the potential to capture the entire complexity of the tumor, which is particularly important for highly heterogeneous or metastatic tumors. Here we report the findings of an analytical performance evaluation of the Illumina TruSight Oncology 500 ctDNA assay, a 523-gene NGS panel developed for ctDNA-based comprehensive genomic profiling (CGP) of tumors, using reference and patient samples. Using 30 ng cfDNA, the assay showed high sensitivity and low variant detection variability for SNVs, indels, and fusions down to a VAF of 0.5% in the reference samples and VAFs, which were highly concordant with previous ddPCR results in the patient samples. At reduced input amounts (20, 15, and 5 ng) and below VAFs of 0.5%, sensitivity was considerably lower and variant detection variability increased. Covering 523 tumor-associated genes, the assay demonstrated a convincing performance comparable to NGS-based ctDNA assays with smaller gene panels, highlighting its value to screen large numbers of different genes.