The investigation of a pleural effusion is, in general, a very straight forward process with the combination of clinical history, examination, radiology and pleural fluid analysis leading to diagnosis in most cases. While most fluid samples are sent for routine analysis including protein, LDH, glucose, cytology and microbiology, there are a number of more unusual fluid analyses available which in some cases directly lead to, and in others are suggestive of the diagnosis. Moreover, other fluid markers are constantly being evaluated as a diagnostic tool. In this review, we describe these non-routine pleural fluid analyses in detail. English language publications in MEDLINE and references from relevant articles from January 1 1990 to August 1 2009 were reviewed. Keywords searched in combination were pleural fluid, effusion, analysis, transudate, exudate and diagnosis.