Photosynthetic activity is established during chloroplast biogenesis. In this study we used 680 nm red light to overexcite Photosystem II and disrupt photosynthesis in two conditional mutants (var2 and abc1k1) which reversibly arrested chloroplast biogenesis. During biogenesis, chloroplasts import most proteins associated with photosynthesis. Some of these must be inserted in or transported across the thylakoid membrane into the thylakoid lumen. They are synthesized in the cytoplasm with cleavable targeting sequences and the lumenal ones have bi-partite targeting sequences (first for the chloroplast envelope, second for the thylakoid membrane). Cleavage of these peptides is required to establish photosynthesis and a critical step of chloroplast biogenesis. We employ a combination of Western blotting and mass spectrometry to analyze proteins in var2 and abc1k1. Under red light, var2 and abc1k1 accumulated incompletely cleaved processing intermediates of thylakoid proteins. These findings correlated with colorless cotyledons, and defects in both chloroplast morphology and photosynthesis. Together the results provide evidence for the requirement of active photosynthesis for processing of photosystem-associated thylakoid proteins and concomitantly progression of chloroplast biogenesis.