Purpose <p>In recent decades, the market demand of recombinant human erythropoietin (rHuEPO) has been increased owing to its wide range of applications. The ability of rHuEPO products and biosimilars to stimulate erythropoiesis has been commercially explored for treatment of various types of anemic patients including those suffering from comorbidities such as acute renal disease, HIV or cancer. The regulation of therapeutic effectiveness of these rHuEPO biosimilars is enforced by potency assay using in vivo methods as described in compendia.</p> Methods <p>Several researchers have demonstrated the Substitution of animal based methods with in vitro methods like cell-based bioassays, immunoassays, colorimetric methods, etc. to comply with 3Rs policy alongwith the developing more sensitive consistent and specific potency estimation test for rHuEPO.</p> Results <p>Utilization of animal-based studies for estimation of potency parameter of products presents several challenges which necessitates the need of a paradigm shift to in vitro assays that are much more consistent, high throughput, ethical and reliable. Though this shift requires establishment of a validated method that effectively reflects the in vivo-in vitro correlation.</p> Conclusion <p>This review will summarize current potency estimation methods, the lacuna and challenges in their application and the suitable non-animal-based substitution methods.</p>