Life sciences faculty agree that developing scientific literacy is an integral part of undergraduate education and report that they teach these skills. However, few measures of scientific literacy are available to assess students' proficiency in using scientific literacy skills to solve scenarios in and beyond the undergraduate biology classroom. In this paper, we describe the development, validation, and testing of the Test of Scientific Literacy Skills (TOSLS) in five general education biology classes at three undergraduate institutions. The test measures skills related to major aspects of scientific literacy: recognizing and analyzing the use of methods of inquiry that lead to scientific knowledge and the ability to organize, analyze, and interpret quantitative data and scientific information. Measures of validity included correspondence between items and scientific literacy goals of the National Research Council and Project 2061, findings from a survey of biology faculty, expert biology educator reviews, student interviews, and statistical analyses. Classroom testing contexts varied both in terms of student demographics and pedagogical approaches. We propose that biology instructors can use the TOSLS to evaluate their students' proficiencies in using scientific literacy skills and to document the impacts of curricular reform on students' scientific literacy. (Contains 4 figures and 6 tables.)