Summary PESTO is a widely applicable and highly customizable toolbox for parameter estimation in MathWorks M ATLAB. It offers scalable algorithms for optimization, uncertainty and identifiability analysis, which work in a very generic manner, treating the objective function as a black box. Hence, PESTO can be used for any parameter estimation problem, for which the user can provide a deterministic objective function in MATLAB. Availability and implementation PESTO is a MATLAB toolbox, freely available under the BSD license. The source code, along with extensive documentation and example code, can be downloaded from https://github.com/ICB-DCM/PESTO/. Contact firstname.lastname@example.org Supplementary informationSupplementary dataare available at Bioinformatics online.