Accurate COVID-19 diagnosis is critical and although PCR is highly sensitive, false negative results do occur. To investigate suboptimal sample collection as a possible cause, we quantified human DNA levels, hypothesizing that could serve as a stable molecular marker. We identified 40 suspected false negative nasopharyngeal swab test results, 23 negative samples from individuals with a positive test within ±12 days and 17 samples from individuals who tested negative but had a high clinical suspicion. We began by validating a SARS-CoV-2-nested RT-PCR sequencing protocol targeting ORF1a and Spike on SARS-CoV-2-negative and positive samples. The in-house lower limit of detection of the nested RT-PCR assay was ~1.5 copies/reaction, which is lower than the reported LLOD. All suspect false-negative samples again tested negative. Then, we investigated human DNA. We used a multiplexed ddPCR protocol for absolute human RPP30 gene copy number quantification and observed significantly lower human DNA levels in the suspected false negative samples Further stratification revealed that the samples harbored less human DNA compared with the control group. This supports suboptimal biological sampling as a contributing cause of false negative COVID-19 test results. Our results underscore the importance of a proper technique in the collection of high-quality nasopharyngeal specimens.