Current treatment options for uterine fibroids are limited to hormonal manipulation or surgical intervention. We aimed to develop an in-vitro model to mirror collagen deposition and extracellular matrix (ECM) formation, the principal features of uterine fibroids, to enable testing of novel therapeutics. Macromolecular crowding with Ficoll 400 and Ficoll 70 in cultures of human uterine myometrial smooth muscle cells containing ascorbic acid, provided the basis for this model. These culture conditions mimic the ‘crowded’ nature of the in-vivo extracellular environment by incorporating neutral, space-filling macromolecules into conventional cell cultures. This method of culture facilitates appropriate ECM deposition, thus closely representing the in-vivo fibrotic phenotype of uterine fibroids. Macromolecular crowding in Ficol cultures containing ascorbic acid reduced myometrial smooth muscle cell proliferation and promoted collagen production. Under these conditions, collagen was processed for extracellular deposition as demonstrated by C-propeptide cleavage from secreted procollagen. The fibrosis marker activin, was increased relative to its natural inhibitor, follistatin, in crowded culture conditions while addition of exogenous follistatin reduced collagen (Col1A1) gene expression. This in-vitro model represents a promising development for the testing of therapeutic interventions for uterine fibroids. However, it does not recapitulate the full in-vivo pathology which can include specific genetic and epigenetic alterations that have not been identified in the myometrial smooth muscle (hTERT-HM) cell line. Following screening of potential therapeutics using the model, the most promising compounds will require further assessment in the context of individual subjects including those with genetic changes implicated in fibroid pathogenesis.