Minimal cells represent a transformative approach in synthetic biology, which aims to create simple autonomous living organisms by removing the nonessential genes and preserving essential genes required for cellular function. This chapter analyses the development, methodologies, and applications of minimal cells, which primarily focuses on two approaches: the top-down approach which involves sequentially removal of the nonessential genes from the cells and the bottom-up approach, which designs the whole biological system from scratch using essential components that can be used for cell survival, metabolism, and functional activities. The chapter also highlights the considerable achievements in minimal cell engineering, specifically the development of JCVI-Syn series (Syn1.0, Syn2.0, and Syn3.0) through sequential removal of genes, which leads to reduce genome size from 1078 kb to 531 kb, that represents the successful implementation of minimal cells. With the help of Tn5 transposon mutagenesis, researchers classified genes under essential (E), quasi-essential (QE), and nonessential (NE) genes. The JCVI-Syn3.0 minimal cell consists of approximately 473 genes, which demonstrates that cellular life can be sustained with significantly reduced genomic complexity. Model species, such as Escherichia coli and Bacillus subtilis have served for genome minimization studies, achieving 20–35% genome size reduction while maintaining cellular viability. These reduced genomes lead to improve energy efficiency, while some strains also showed 50% faster growth rates in comparison with the wild-type strain due to removal of NE genes, which may reduce the growth rate by more energy consumption. Moreover, challenges such as epistatic effects and synthetic lethality highlight the complexity of gene interactions and the need for sophisticated approaches beyond simple gene deletion. Minimal cells can be applied in biotechnology, therapeutic platforms, and fundamental biological research. Their simplified genomes make them ideal hosts for synthetic circuits, drug delivery systems, and biosensing applications. Additionally, minimal cells provide unique insights into the origin of life by identifying the minimal genetic requirements for autonomous cellular function.