Matrix-assisted laser desorption/ionization imaging mass spectrometry was used to analyze donor eye tissue specimens for phospholipid content to evaluate lipid distribution. Phosphatidylcholines and sphingomyelins were detected in the positive ion mode using 2,5-dihydroxybenzoic acid as the matrix. During this study, unknown ion signals in the lower m/z region (less than m/z 400) were detected, mainly in the far periphery of human flat-mounted tissue but not in age-matched rhesus monkey tissue prepared in a similar manner. The unknown ion signals occurred at m/z 304, 332, 360, and 388. These ions were subjected to tandem mass spectrometry directly from the tissue sample, and exact mass measurements of extracts were prepared for further identification. These ions were identified as alkyl dimethylbenzylammonium surfactants (benzalkonium chlorides (BACs)). The classification of these species was verified by comparing an eye tissue extract to an over-the-counter eye-care product containing BACs.