Since several decades, secondary ion mass spectrometry (SIMS) coupled to time of flight (TOF) is used for atomic or small inorganic/organic fragments imaging on different materials. With the advent of polyatomic ion sources leading to a significant increase of sensitivity in combination with a reasonable spatial resolution (1–10 μm), TOF-SIMS is becoming a more and more popular analytical platform for MS imaging. Even if this technique is limited to small molecules (typically below 1,000 Da), it offers enough sensitivity to detect and locate various classes of lipids directly on the surface of tissue sections. This chapter is thus dedicated to the TOF-SIMS analysis of lipids in positive and negative ion modes on rat brain tissue sections using a bismuth cluster ion source.