•The Alinity m and NeuMoDx 288 are fully-automated molecular sample-in-result-out systems.•The Alinity m and NeuMoDx HCV assays showed good comparability.•The HBV assay of the NeuMoDx seems to under-quantify compared to the Alinity m assay.•Qualitative differences of the Alinity m and NeuMoDx HIV-1 and HBV assays were most apparent in samples with lower viral loads. Viral load (VL) determination in patients with human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) is essential for proper patient management and follow-up. New molecular platforms have been developed to fully automate these diagnostic assays. Evaluation of the clinical performance of HIV-1, HBV and HCV VL assays on the Alinity m (Abbott) and NeuMoDx (Qiagen) molecular platforms. Test panels of the three viruses have been compiled of 100 plasma and/or serum samples per target containing non-detectable, non-quantifiable and quantifiable VLs. All samples were retrospectively tested on the Alinity m and NeuMoDx platforms according to manufacturers’ instructions. A total of 74, 86 and 66 samples with valid results for both platforms were included in the HIV-1, HBV and HCV analysis respectively. Overall qualitative agreement of the assays on both platforms was 78% for HIV-1, 93% for HBV and 100% for HCV. Quantitative agreement (less than 0.5 log difference) was shown to be 68% for HIV-1, 68% for HBV and 94% for HCV. The Alinity m and NeuMoDx HCV assay have a comparable performance. Quantification differences in the HIV-1 assay were mostly apparent in the lower VLs and under-quantification of the NeuMoDx HBV assay was observed.