PURPOSE: Isolation of primary human hepatocytes (PHH) from liver specimens typically relies on the two-step perfusion method, which requires large samples, substantial resources, specialised expertise and suitable vessels for cannulation. Although non-perfusion methods exist, they yield low numbers of hepatocytes and inadequately assess hepatocyte purity. We compared and optimised these methods to develop an improved technique that isolates high yields of viable PHH from non-perfusable liver specimens. METHOD: In the optimised protocol, non-cancerous resected liver tissue (mean weight: 8.5 ± 2.0 g, SEM) was sliced into 350 μm sections using a vibratome and subjected to a two-step isolation digestion with ethylenediaminetetraacetic acid (EDTA) and collagenase. Cell yield and viability were assessed using propidium iodide staining. Cell populations were characterised by immunofluorescent imaging. RESULTS: The optimised protocol yielded 1.17 ± 0.2 × 106 viable PHH per gram of tissue, approximately 2-fold higher than other non-perfusion protocols, although lower than yields reported for perfusion protocols in the literature. Notably, our protocol achieved an average hepatocyte viability of 80 ± 4%, which surpassed the reported average for published non-perfusion methods. Staining for glycogen and albumin secretion confirmed the functional integrity of the isolated PHH. The protocol was also effective with steatotic liver tissue, yielding 1.0 ± 0.1 × 10⁶ viable PHH per gram with 83 ± 2% viability. Most liver specimens were obtained from patients who had undergone neoadjuvant chemotherapy, however, no trend related to chemotherapy treatment was observed. CONCLUSIONS: Our non-perfusion protocol permits the isolation of viable and functional PHH from a diverse range of liver samples. This advancement provides a practical alternative to perfusion methods and will extend the use of PHH in research and drug development. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s44164-025-00097-4.