Macrocyclic natural products have been recognized and utilized as new platforms for designing drugs in pharmaceutical research because the constrained three-dimensional shapes and the large surface areas of these complex structures enable selective binding to conventionally undruggable targets. Since natural products are not necessarily ideal for medicinal use, structural optimization is required to obtain superior drug candidates. However, the modifications to macrocyclic natural products that will afford the best pharmacological characteristics cannot be known a priori. Therefore, this optimization procedure requires the exploration of a vast array of natural product analogues to identify new compounds with more desirable properties. To fully explore the chemical space around complex macrocyclic natural products, the construction of a large number of analogues is required. In this review, we provide an overview of the efficient synthetic construction of the analogues of macrocyclic natural products and the evaluation of their biological activities. The examples of the comprehensive structure-activity relationship (SAR) studies depicted herein led to the discoveries of biologically useful analogues. These studies illustrate the importance of designing building blocks and coupling strategies to synthesize a variety of natural product based analogues.