Abstract. We present a major new release of ASSA-PBN, a software tool for modelling, simulation, and analysis of probabilistic Boolean networks (PBNs). PBNs are a widely used computational framework for modelling biological systems. The steady-state dynamics of a PBN is of special interest and obtaining it poses a significant challenge due to the state space explosion problem which often arises in the case of large biological systems. In its previous version, ASSA-PBN applied efficient statistical methods to approximately compute steady-state probabilities of large PBNs. In this newly released version, ASSA-PBN not only speeds up the computation of steady-state probabilities with three different realisations of parallel computing, but also implements parameter estimation and techniques for in-depth analysis of PBNs, i.e., influence and sensitivity analysis of PBNs. In addition, a graphical user interface (GUI) is provided for the convenience of users.