Author Summary Muscle functionality relies on the correct assembly of myofibrils, which are composed of tandem arrays of basic functional contractile units called the sarcomeres. Many mutations in genes encoding sarcomeric proteins cause muscle diseases such as congenital myopathy and dilated cardiac hypertrophy. Understanding the process of sarcomere assembly is not only relevant to the understanding of how protein complexes interact to form complex supra-molecular structures, but also of great significance to medicine for muscle diseases. Here, by taking advantage of our newly developed primary muscle cell culture method, we reevaluate sarcomere assembly by systematically analyzing the functional relationship of sarcomeric proteins using RNA interference or genetic ablation techniques. Our analysis leads us to propose a “two-state” model whereby sarcomeric proteins exist either in the “chaotic” state with independently assembled differential functional complexes or the “highly ordered suprastructure” state made from these complexes. Because we fail to detect any previously hypothesized sarcomere assembly intermediates in our system, our data support the model that sarcomere assembly is a highly coordinated process mediated by multiple latent protein complexes and does not occur in a step-wise fashion.