Lenacapavir (LEN) is a first-in-class capsid inhibitor (CAI) that targets multiple stages of the HIV-1 lifecycle, showing efficacy in heavily treatment-experienced (HTE) individuals with multidrug-resistance (MDR) and in pre-exposure prophylaxis (PrEP). This study aimed to characterize a novel home-made next-generation sequencing (NGS) protocol targeting HIV-1 capsid (CA) region using both HIV-1 RNA from plasma and HIV-1 DNA from peripheral-blood-mononuclear-cells (PBMCs). Accordingly, 60 samples (41 HIV-1 RNA, 19 HIV-1 DNA) with various HIV-1 subtypes and viremia levels were tested. Molecular amplification was successful in 83.3% of cases (75.6% HIV-1 RNA and 100% HIV-1 DNA), with sequencing achieved in 92.0% of amplified samples (87.1% HIV-1 RNA and 100% HIV-1 DNA). The protocol showed high NGS performance with HIV-1 RNA samples with viremia >500 copies/mL (92.6%) and a slight impact of subtype-associated variability (subtype B: amplification and sequencing 86.0% and 91.9%; non-B: 76.5% and 92.3%, respectively). Reproducibility was fully confirmed by pairwise similarity analyses at 10% and 20% frequency cutoff, upon reprocessing 13 HIV-1 RNA samples. This protocol provides an important tool for personalized HIV-1 treatment with CAI-based strategies, enabling efficient characterization of LEN resistance mutations in the CA region across different HIV-1 subtypes, using both DNA and RNA samples.