Angiogenesis research has grown extensively over the past few decades with the recognition that angiogenesis plays a key role in embryonic development, wound healing, and also pathological conditions such as tumor metastasis. Numerous in vitro angiogenesis assays have been designed to mimic in vivo angiogenesis process. The in vitro assays are quick, simple, quantitative, and cost-effective, and have thereby played valuable roles in evaluation of the effects of test compounds on angiogenesis. However, each in vitro assay represents an individual step of a complex angiogenic cascade. Furthermore, significant diversities exist within endothelial cell types and within basement membrane extracts used in the assays. Thus, choosing appropriate cell types and assay conditions that most closely resemble the disease-specific angiogenesis being studied is essential to obtain meaningful results. Performing multiple in vitro assays together should also be considered to get data that is translatable from the preclinical to the clinical stage. For these reasons, increasing our understanding of in vitro assays is necessary for proper assay performance as well as correct interpretation of the results. Here we discuss two main critical factors regarding endothelial cell types and basement membrane extracts that are known to largely affect in vitro angiogenesis assays. We also introduce recent improvements in the most widely-used in vitro angiogenesis assays, which are 2-dimensional (2D) tube formation assays and 3-dimensional (3D) spheroid angiogenesis assay.