This study investigated the nature of pre-service science teachers' (PSTs') situation-specific skills for informal formative assessment (FA) and their awareness of activating these skills in classroom-like situations. Video-based interviews were conducted to collect data from fifteen PSTs before they participated in a teacher education program. Authentic classroom videos capturing evidence of student thinking were used as probes to elicit information on the PSTs' perceptions, interpretations, and decision-making in various classroom-like situations that afforded opportunities for FA. The findings indicate that the PSTs tended to focus on content-generic evidence and the responses of students in the whole class, to infer student thinking at a non-content specific level, and to propose responses that were vague or devoid of scientific content. On re-exposure to the same evidence of student thinking captured in short video clips, and with more specific prompting questions, more of the PSTs identified content-specific evidence from individual students and interpreted the nuances of student thinking at a content-specific level. Yet, the capacity of the PSTs to propose student-focused instructional moves remained limited. Overall, the findings suggest that the PSTs were not particularly aware of applying their situation-specific skills for informal FA when making on-the-fly teaching decisions in classroom-like situations that offer rich opportunities for FA. This study adds to the existing understanding of PST knowledge/skills for informal FA and provides a mechanism for eliciting and characterizing teachers' situation-specific skills for informal FA. The implications for initial teacher education are discussed.