The Neutral Theory of Molecular Evolution is considered the most powerful theory to predict the evolutionary behavior of proteins. One of the main predictions of this theory is that essential proteins should evolve slower than dispensable ones owing to increased selective constraints. Comparison of genomes of different species, however, have revealed only small differences between the rates of evolution of essential and non-essential proteins. It has been argued that comparing relatively distant genomes may entail a number of limitations. For instance, many of the genes that are dispensable in controlled lab conditions may be essential in some of the conditions faced in nature. Moreover, essentiality can change during evolution, and rates of protein evolution are simultaneously shaped by a variety of factors, whose individual effects are difficult to isolate. Here, we conducted two parallel mutation accumulation experiments in Escherichia coli, during 5500–5750 generations, and compared the genomes at different points of the experiments. Our approach (a short-term experiment, under highly controlled conditions) enabled us to overcome many of the limitations of previous studies. We observed that essential proteins evolved significantly slower than non-essential ones during our experiments. Strikingly, rates of protein evolution were not affected by expression level, protein abundance, or protein length.