Model organisms such as the Gram-positive bacterium Bacillus subtilis have been studied intensively for decades. However, even for such model organisms no function has been identified for about one fourth of all proteins. It has recently been realized that such understudied proteins as well as poorly studied functions set a limitation to our understanding of the requirements for cellular life, and the Understudied Proteins Initiative has been launched. Obviously, poorly studied proteins that are strongly expressed, are likely to be most important to the cell and should therefore have priority in further studies. Since the functional analysis of unknown proteins can be extremely laborious, a minimal knowledge is required prior to targeted functional studies. In this review, we discuss strategies to obtain such a minimal annotation, e.g. from global interaction, expression or localization studies. We present a set of 41 highly expressed and poorly studied proteins of B. subtilis. Several of these proteins are thought or known to bind RNA and/or the ribosome, some may may control the metabolism of B. subtilis, and another subset of particularly small proteins may act as regulatory elements to control the expression of downstream genes. Moreover, we discuss the challenges of poorly studied functions with a focus on RNA-binding proteins, amino acid transport and the control of metabolic homeostasis. The identification of the functions of the selected proteins will strongly advance our knowledge on B. subtilis, but also on other organisms since many of the proteins are conserved in many groups of bacteria.